Exogenous ketone supplements for reducing anxiety-related behavior

ABSTRACT

Methods of treating anxiety disorders or reducing anxiety-related behaviors. The methods include administering a therapeutically effective amount of ketone supplementation, such as butanediol, ketone esters (e.g., 1,3-butanediol-acetoacetate diester) and/or ketone salts (e.g., beta-hydroxybutyrate-mineral salt), chronically, sub-chronically, or acutely, with or without admixture with a medium chain triglyceride or in combination. It was determined herein that ketone supplementation reduced anxiety in rats on elevated plus maze as measured by less entries to closed arms, more time spent in open arms, more distance travelled in open arms, and delayed latency to entrance to closed arms, when compared to control. Along with reducing anxiety-related behavior, the chronic, sub-chronic, and acute ketone supplements also caused significant elevation of blood βHB levels and changed blood glucose levels.

CROSS-REFERENCE TO RELATED APPLICATIONS

This nonprovisional application claims priority to U.S. ProvisionalPatent Application No. 62/289,749, entitled “Exogenous KetoneSupplements for Reducing Anxiety-Related Behavior”, filed Feb. 1, 2016by the same inventors, the entirety of which is incorporated herein byreference.

RELATED APPLICATION

This application relates to International Patent Application No.PCT/US2014/031237, entitled “Compositions and Methods for ProducingElevated and Sustained Ketosis”, filed Mar. 19, 2014 by several of thecurrent inventors, the entirety of which is incorporated herein byreference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant Nos.N00014-09-1-0244 and N00014-13-1-0062 awarded by the Office of NavalResearch. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION 1. Field of the Invention

This invention relates, generally, to therapies for anxiety-relatedbehavior. More specifically, it relates to short- and long-term ketosisto treat and/or prevent anxiety-related behavior.

2. Brief Description of the Prior Art

Anxiety

Anxiety disorders, such as generalized anxiety disorder, phobias, andpanic disorder, are the most prevalent type of mental disorders (Li,Xiaobai Using the conditioned fear stress (CFS) animal model tounderstand the neurobiological mechanisms and pharmacological treatmentof anxiety. Shanghai Archives of Psychiatry, 2012, Vol. 24, No. 5241-249). Anxiety can be associated with psychiatric morbidity,disability, increased healthcare utilization, and mortality in thegeneral population (Teri, L., et al. 1999. ‘Anxiety in Alzheimer'sDisease: Prevalence and Comorbidity’. The Journals of Gerontology SeriesA: Biological Sciences and Medical Sciences 54 (7): M348-M352.doi:10.1093/gerona/54.7.m348). These symptoms can cause significantdistress interfering with an individual's quality of life. This commonlyoccurs along with other mental or physical illnesses, which may maskanxiety symptoms or even aggravate them. Some symptoms, like fear andworry, occur in all anxiety disorders including generalized anxietydisorders, panic disorder, and social anxiety disorder (Mula, Marco.2013. ‘Treatment of Anxiety Disorders, in Epilepsy: An Evidence-BasedApproach’. Epilepsia 54: 13-18. doi:10.1111/epi.12101).

Knowledge relating to the exact cause and pathomechanism(s) of anxietydisorders is far from complete; however, it is known that the amygdalais determinant in the experience of fear and anxiety by mediating theautonomic and endocrine responses through the output to thehypothalamus, and also avoidance behavior through the output to theperiaqueductal gray matter (Engin E, et al. The effects ofintra-cerebral drug infusions on animals' unconditioned fear reactions:a systematic review. Prog Neuropsychopharmacol Biol Psychiatry. 2008Aug. 1; 32(6):1399-419; Li, Xiaobai Using the conditioned fear stress(CFS) animal model to understand the neurobiological mechanisms andpharmacological treatment of anxiety. Shanghai Archives of Psychiatry,2012, Vol. 24, No. 5 241-249; Mula, Marco. 2013. ‘Treatment of AnxietyDisorders, in Epilepsy: An Evidence-Based Approach’. Epilepsia 54:13-18. doi:10.1111/epi.12101). Previous studies also show thatserotonergic, glutamatergic, and GABAergic system have roles in theregulation of anxiety (Nagy J, et al. Anti-anxiety action of diazepamafter intra-amygdaloid application in the rat. Neuropharmacology. 1979;18(6): 573-576; Kakui N, et al. Anxiolytic-like profile of mirtazapinein rat conditioned fear stress model: Functional significance of5-hydroxytryptamine 1A receptor and alpha1-adrenergic receptor.Pharmacol Biochem Behav. 2009 May; 92(3):393-8; Li, Xiaobai Using theconditioned fear stress (CFS) animal model to understand theneurobiological mechanisms and pharmacological treatment of anxiety.Shanghai Archives of Psychiatry, 2012, Vol. 24, No. 5 241-249; Dias B G,et al. Towards new approaches to disorders of fear and anxiety. CurrOpin Neurobiol. 2013 June; 23(3):346-52).

Further, anxiety and depression are common problems affecting peoplewith epilepsy and Alzheimer's disease (AD), and can exacerbate symptomsof GLUT1 deficiency syndrome (GLUT1 DS). Comorbidity between anxiety,depression, and AD has been recognized (Teri, L., et al. 1999. ‘Anxietyin Alzheimer's Disease: Prevalence and Comorbidity’. The Journals ofGerontology Series A: Biological Sciences and Medical Sciences 54 (7):M348-M352. doi:10.1093/gerona/54.7.m348; Hamid H, et al. (2011) Anxietysymptoms in epilepsy: salient issues for future research. Epilepsy Behav22:63-68; Mula, Marco. 2013. ‘Treatment of Anxiety Disorders, inEpilepsy: An Evidence-Based Approach’. Epilepsia 54: 13-18.doi:10.1111/epi.12101), and anxiety plays a key role in suicidalityamong patients with depression (Placidi G P A, et al. (2000) Anxiety inmajor depression: relationship to suicide attempts. Am J Psychiatry157:1614-1618). Interestingly, the same brain regions involved in asignificant proportion of patients with focal epilepsy, such as theamygdala and the hippocampus, also play a key role in the neurobiologyof anxiety (Li, Xiaobai Using the conditioned fear stress (CFS) animalmodel to understand the neurobiological mechanisms and pharmacologicaltreatment of anxiety. Shanghai Archives of Psychiatry, 2012, Vol. 24,No. 5 241-249; Dias B G, et al. Towards new approaches to disorders offear and anxiety. Curr Opin Neurobiol. 2013 June; 23(3):346-52).

Ketones

Ketones are produced naturally in the liver only under certainphysiological conditions associated with the suppression of the hormoneinsulin: starvation, fasting, calorie restriction, prolonged exercise,or during the consumption of high fat, low carbohydrate KD. Therestrictive nature of these states has limited the clinicalapplicability of therapeutic ketosis due to practical considerations. Inan effort to circumvent this dilemma, researchers have recentlydeveloped a number of exogenous ketogenic supplements, ketogenicprecursors that are metabolized to produce a dose-dependent elevation ofβHB and AcAc in the blood (Clarke, K., Tchabanenko, K., Pawlosky, R.,Carter, E., Todd, K. M., Musa-Veloso, K., Ho, M., Roberts, A.,Robertson, J., Vanitallie, T. B., Veech, R. L. (2012) Kinetics, safetyand tolerability of (R)-3-hydroxybutyl (R)-3-hydroxybutyrate in healthyadult subjects. Regul Toxicol Pharmacol. 63(3):401-8; Veech, R. (2004).The therapeutic implications of ketone bodies: the effects of ketonebodies in pathological conditions: ketosis, ketogenic diet, redoxstates, insulin resistance, and mitochondrial metabolism.Prostaglandins, leukotrienes, and essential fatty acids 70, 309-319;Kesl, S. L., Poff, A. M., Ward, N. P., Fiorelli, T. N., Ari, C., VanPutten, A. J., Sherwood, J. W., Arnold, P., D'Agostino, D. P. (2016)Effects of exogenous ketone supplementation on blood ketone, glucose,triglyceride, and lipoprotein levels in Sprague-Dawley rats. Nutr Metab(Lond). 4; 13:9). Induction of hyperketonemia produces acute and chronicchanges in metabolic physiology and neuropharmacological pathways thatprovide therapeutic effects in varied disease states.

Previous studies have demonstrated the use of exogenous ketones as ameans to induce a dose-dependent hyperketonemia (1-7 mM) in rats, mice,dogs, pigs, and humans (Brunengraber, H. (1997) Potential of ketone bodyesters for parenteral and oral nutrition. Nutrition. 13(3):233-5;Ciraolo, S. T., Previs, S. F., Fernandez, C. A., Agarwal, K. C., David,F., Koshy, J., Lucas, D., Tammaro, A., Stevens, M. P., and Tserng, K. Y.(1995). Model of extreme hypoglycemia in dogs made ketotic with(R,S)-1,3-butanediol acetoacetate esters. Am J Physiol 269, 75; Clarke,K., Tchabanenko, K., Pawlosky, R., Carter, E., Todd, K. M., Musa-Veloso,K., Ho, M., Roberts, A., Robertson, J., Vanitallie, T. B., Veech, R. L.(2012) Kinetics, safety and tolerability of (R)-3-hydroxybutyl(R)-3-hydroxybutyrate in healthy adult subjects. Regul ToxicolPharmacol. 63(3):401-8; Desrochers, S., David, F., Garneau, M., Jetté,M., and Brunengraber, H. (1992). Metabolism of R- and S-1,3-butanediolin perfused livers from meal-fed and starved rats. J Biochem 285 (Pt 2),647-653; Puchowicz, M. A., Smith, C. L., Bomont, C., Koshy, J., David,F., Brunengraber, H. (2000) Dog model of therapeutic ketosis induced byoral administration of R,S-1,3-butanediol diacetoacetate. J NutrBiochem. 11(5):281-7. PubMed PMID: 10876102; Srivastava, S., Kashiwaya,Y., King, M., Baxa, U., Tam, J., Niu, G., Chen, X., Clarke, K., andVeech, R. (2012). Mitochondrial biogenesis and increased uncouplingprotein 1 in brown adipose tissue of mice fed a ketone ester diet. FASEBJournal: Official publication of the Federation of American Societiesfor Experimental Biology 26, 2351-2362; Desrochers, S., Quinze, K.,Dugas, H., Dubreuil, P., Bomont, C., David, F. et al. (1995).R,S-1,3-butanediol acetoacetate esters, potential alternates to lipidemulsions for total parenteral nutrition. Journal Nutr Biochem, 6(2),111-118). Exogenous ketogenic supplementation mimics the metabolic andphysiologic effects of the KD, including enhancing mitochondrialbiogenesis, anaplerosis, suppression of glycolysis, and increasing ATPand adenosine production, all thought to mediate the therapeutic effectsof KD in epilepsy (Kesl, S., Prather, J., Sherwood, J., Gould, L.,D'Agostino, P. D. (2014). Sustaining dietary ketosis to improve bloodflow and wound healing in young and aged Fisher rats. The FASEB Journal28 no. 1 Supplement 734.7, 73; Poff, A. M., Ward, N., Seyfried, T. N.,Arnold, P., D'Agostino, D. P. (2015). Non-Toxic Metabolic Management ofMetastatic Cancer in VM Mice: Novel Combination of Ketogenic Diet,Ketone Supplementation, and Hyperbaric Oxygen Therapy. PLoS One 10:e0127407, 125; Srivastava, S., Kashiwaya, Y., King, M., Baxa, U., Tam,J., Niu, G., Chen, X., Clarke, K., and Veech, R. (2004). The therapeuticimplications of ketone bodies: the effects of ketone bodies inpathological conditions: ketosis, ketogenic diet, redox states, insulinresistance, and mitochondrial metabolism. Prostaglandins, leukotrienes,and essential fatty acids 70, 309-319). Mitochondrial biogenesis andincreased uncoupling protein 1 in brown adipose tissue of mice fed aketone ester diet. FASEB Journal: Official publication of the Federationof American Societies for Experimental Biology 26, 2351-2362; Veech,2004).

Anecdotal reports suggest that nutritional ketosis can promote areduction in anxiety, although there is currently no convincing evidenceto indicate that elevated ketone levels would reduce anxiety in humans(Ehrenreich M J. A case of the re-emergence of panic and anxietysymptoms after initiation of a high-protein, very low carbohydrate diet.Psychosomatics. 2006 March-April; 47(2):178-9 Engin E, et al. Theeffects of intra-cerebral drug infusions on animals' unconditioned fearreactions: a systematic review. Prog Neuropsychopharmacol BiolPsychiatry. 2008 Aug. 1; 32(6):1399-419). Nutritional ketosis is a statewhen the body utilizes ketones instead of glucose as the primarymolecule of energy metabolism (Veech, R. (2004) The therapeuticimplications of ketone bodies: the effects of ketone bodies inpathological conditions: ketosis, ketogenic diet, redox states, insulinresistance, and mitochondrial metabolism. Prostaglandins, leukotrienes,and essential fatty acids 70, 309-319). Specifically, the body increasesconversion of ketones such as beta-hydroxybutyrate (βHB) into acetyl-CoAand thus generates ATP (Yudkoff M, et al. The ketogenic diet and brainmetabolism of amino acids: relationship to the anticonvulsant effect.Annu Rev Nutr. 2007; 27:415-30). This can be achieved by fasting orfollowing a ketogenic diet (KD), which is a high-fat (70-85% of dailycaloric intake) and low-carbohydrate (5-10%) diet (Kwiterovich, P., etal. (2003) Effect of a high-fat ketogenic diet on plasma levels oflipids, lipoproteins, and apolipoproteins in children. JAMA: the journalof the American Medical Association 290, 912-920; De Giorgis, Valentina,et al. 2013. ‘GLUT1 Deficiency Syndrome 2013: Current State of The Art’.22 (10): 803-811. doi:10.1016/j.seizure.2013.07.003).

Maintaining long-term ketosis has proven to be beneficial in epilepticpatients by decreasing the frequency and severity of seizures (Kossoff EH, et al. Ketogenic diets: treatments for epilepsy and other disorders.5th ed. New York N.Y.: Demos Medical; 2011). Nutritional ketosis alsohas confirmed beneficial effects in patients with AD, GLUT1 DS, andcancer (Vegiotti and De Giorgis, 2014; Poff, A., et al. (2013) Theketogenic diet and hyperbaric oxygen therapy prolong survival in micewith systemic metastatic cancer. PloS one 8; Poff, A. M., et al. (2014)Ketone supplementation decreases tumor cell viability and prolongssurvival of mice with metastatic cancer. International journal ofcancer). GLUT1 DS results from impaired glucose transport into the brain(Klepper J, et al. Facilitated glucose transporter protein type 1(GLUT1) deficiency syndrome: impaired glucose transport into brain—areview. Eur J Pediatr. 2002 June; 161(6):295-304. Epub 2002 Apr. 16.Review. PubMed PMID:12029447); however, ketones use another transporterto enter the central nervous system (CNS), providing an alternativesource of fuel. Therefore, nutritional ketosis is used as a treatmentoption in GLUT1 patients (De Giorgis, Valentina, et al. 2013. ‘GLUT1Deficiency Syndrome 2013: Current State of The Art’. 22 (10): 803-811.doi:10.1016/j.seizure.2013.07.003), effectively correcting the impairedbrain energy metabolism and reducing the frequency of the seizures (LeenW G, et al. Glucose transporter-1 deficiency syndrome: the expandingclinical and genetic spectrum of a treatable disorder. Brain. 2010March; 133(Pt 3):655-70. doi:10.1093/brain/awp336. Epub 2010 Feb. 2.PubMed PMID: 20129935).

Additionally, previous studies have shown an anxiolytic effect in theEPM when the antidepressant/antipanic drug phenelzine, agonists and/orantagonists of different neurotransmitter systems (e.g., GABAergic andglutamatergic system) were given acutely to rats (Paslawski T, et al.The antidepressant drug phenelzine produces antianxiety effects in theplus-maze and increases in rat brain GABA. Psychopharmacology (Berl).1996 September; 127(1):19-24. PubMed PMID: 8880939; Engin E, et al. Theeffects of intra-cerebral drug infusions on animals' unconditioned fearreactions: a systematic review. Prog Neuropsychopharmacol BiolPsychiatry. 2008 Aug. 1; 32(6):1399-419). Additionally, the anxiolyticproperties of cannabidiol were tested in an EPM in rats, and itsignificantly increased the entry ratio (open/total number of entries),suggesting an anxiolytic-like effect (Guimarães F S, et al. Anxiolyticeffect in the elevated plus-maze of the N/MDA receptor antagonist AP7microinjected into the dorsal periaqueductal grey. Psychopharmacology(Berl). 1991; 103(1):91-4; Gomes F V, et al. The anxiolytic-like effectsof cannabidiol injected into the bed nucleus of the stria terminalis aremediated by 5-HT1A receptors. Psychopharmacology (Berl). 2011 February;213(2-3):465-73. doi: 10.1007/s00213-010-2036-z. Epub 2010 Oct. 14.PubMed PMID: 20945065).

It has been demonstrated that KD may (i) decrease extracellularglutamate release/level by means of inhibition of vesicular glutamatetransporter, (ii) increase adenosine level, and (iii) augment theGABAergic effects by GABA_(A) receptors (Yudkoff M, et al. The ketogenicdiet and brain metabolism of amino acids: relationship to theanticonvulsant effect. Annu Rev Nutr. 2007; 27:415-30; Engin E, et al.The effects of intra-cerebral drug infusions on animals' unconditionedfear reactions: a systematic review. Prog Neuropsychopharmacol BiolPsychiatry. 2008 Aug. 1; 32(6):1399-419; Masino S A, et al. Purines andneuronal excitability: links to the ketogenic diet. Epilepsy Res. 2012July; 100(3):229-38; Lutas A, et al. The ketogenic diet: metabolicinfluences on brain excitability and epilepsy. Trends Neurosci. 2013January; 36(1):32-40). It has been demonstrated that serotonergic,glutamatergic and GABAergic system of different brain areas such ashippocampus and/or amygdala have a role in the regulation of anxiety:serotonin (5-hydroxytryptamine, 5-HT) transporters, serotonin receptors(e.g., 5-HT1A), N-methyl-D-aspartate (NMDA) receptors and GABA receptors(e.g., GABA_(A) receptors) are potential targets in the treatment ofanxiety disorders (Nagy J, et al. Anti-anxiety action of diazepam afterintra-amygdaloid application in the rat. Neuropharmacology. 1979; 18(6):573-576; Kakui N, et al. Anxiolytic-like profile of mirtazapine in ratconditioned fear stress model: Functional significance of5-hydroxytryptamine 1A receptor and alpha1-adrenergic receptor.Pharmacol Biochem Behav. 2009 May; 92(3):393-8; Li, Xiaobai Using theconditioned fear stress (CFS) animal model to understand theneurobiological mechanisms and pharmacological treatment of anxiety.Shanghai Archives of Psychiatry, 2012, Vol. 24, No. 5 241-249; Sankar R.GABA(A) receptor physiology and its relationship to the mechanism ofaction of the 1,5-benzodiazepine clobazam. CNS Drugs. 2012 Mar. 1;26(3):229-44; Dias B G, et al. Towards new approaches to disorders offear and anxiety. Curr Opin Neurobiol. 2013 June; 23(3):346-52). It wasconcluded that GABAergic system may have critical role in the modulationof the level of anxiety: increased GABAergic transmission may evokeanxiolytic effect (Engin E, et al. The effects of intra-cerebral druginfusions on animals' unconditioned fear reactions: a systematic review.Prog Neuropsychopharmacol Biol Psychiatry. 2008 Aug. 1; 32(6):1399-419;Li, Xiaobai Using the conditioned fear stress (CFS) animal model tounderstand the neurobiological mechanisms and pharmacological treatmentof anxiety. Shanghai Archives of Psychiatry, 2012, Vol. 24, No. 5241-249). Thus, augmentation of the GABAergic effects by means of KD viaGABA_(A) receptors may evoke a decrease in anxiety level.

It has also been demonstrated that (i) KD may increase extracellularadenosine level (Masino S A, et al. Purines and neuronal excitability:links to the ketogenic diet. Epilepsy Res. 2012 July; 100(3):229-38;Lutas A, et al. The ketogenic diet: metabolic influences on brainexcitability and epilepsy. Trends Neurosci. 2013 January; 36(1):32-40),(ii) inhibition of adenosine receptors (A₁R and A_(2A)R) by means ofcaffeine promotes anxious behavior (Klein E, et al. Anxiogenic effectsof m-CPP in patients with panic disorder: comparison to caffeine'sanxiogenic effects. Biol Psychiatry. 1991 Nov. 15; 30(10):973-84), (iii)A₁R- or A_(2A)R-knockout mice showed anxiogenic-like behaviors(Johansson B, et al. Hyperalgesia, anxiety, and decreased hypoxicneuroprotection in mice lacking the adenosine A1 receptor. Proc NatlAcad Sci USA. 2001 Jul. 31; 98(16):9407-12. Epub 2001 Jul. 24; Ledent C,et al. Aggressiveness, hypoalgesia and high blood pressure in micelacking the adenosine A2a receptor. Nature. 1997 Aug. 14;388(6643):674-8) and (iv) modulation of adenosine receptor activitymight be an effective treatment strategy for patients with anxietydisorders (Kovacs, Z., Dobolyi, A. (2013). Anatomical distribution ofnucleoside system in the human brain and implications for therapy. In:Masino S A, Boison D, editors. Adenosine: a key link between metabolismand brain activity. Springer Science: Business Media, New York. p.621-656) Anatomical distribution of nucleoside system in the human brainand implications for therapy. In: Masino S A, Boison D, editors.Adenosine: a key link between metabolism and brain activity. SpringerScience: Business Media, New York. p. 621-656). In addition, as KD mayevoke decreased extracellular glutamate level (Lutas A, et al. Theketogenic diet: metabolic influences on brain excitability and epilepsy.Trends Neurosci. 2013 January; 36(1):32-40) and NMDA receptorantagonists may have anxiolytic effects (Guimarães, F. S., Carobrez, A.P., De Aguiar, J. C., Graeff, F. G. (1991). Anxiolytic effect in theelevated plus-maze of the NMDA receptor antagonist AP7 microinjectedinto the dorsal periaqueductal grey. Psychopharmacology(Berl).103(1):91-4; Engin E, et al. The effects of intra-cerebral druginfusions on animals' unconditioned fear reactions: a systematic review.Prog Neuropsychopharmacol Biol Psychiatry. 2008 Aug. 1; 32(6):1399-419)KD may exert its alleviating effect on anxiety level via glutamatergicsystem. Indeed, a recent study supports the effect of ketone estersincreasing the brain GABA/Glutamate ratio in an animal model ofAngelman's syndrome (Ciarlone, S. L., Grieco, J. C., D'Agostino, D. P.,Weeber, E. J. (2016) Ketone ester supplementation attenuates seizureactivity, and improves behavior and hippocampal synaptic plasticity inan Angelman syndrome mouse model. Neurobiol Dis. 18; 96:38-46).

However, many patients with epilepsy, AD, and about 20% of GLUT1 DSpatients have difficulties with compliance, or experience a loss ofeffectiveness over time (Veggiotti, et al. 2014. ‘Dietary Treatments andNew Therapeutic Perspective in GLUT1 Deficiency Syndrome’. Curr TreatOptions Neurol 16 (5). doi:10.1007/s11940-014-0291-8). Further, verylittle is known about the link between ketone application-evoked changesin CNS and anxiety disorders.

Attempts have been made and studies have been performed to overcome theforegoing problems, though none have been completely effective. Examplesinclude U.S. Patent Application Publication Serial No. 2015/0132280;U.S. Patent Application Publication Serial No. 2015/0344413; YoshihiroKashiwaya et al., “A ketone ester diet exhibits anxiolytic andcognition-sparing properties, and lessens amyloid and tau pathologies ina mouse model of Alzheimer's disease”, Neurobiology of Aging 34 (2013)1530-1539; and Carl E. Strafstrom et al., “The ketogenic diet as atreatment paradigm for diverse neurological disorders”, Frontiers inPharmacology: Neuropharmacology 3(59): 1-8 (April 2012). However, it isstill unknown how to maintain the desired long-term ketosis, both forreducing anxiety-related behavior and for sustaining nutritionalketosis.

Accordingly, what are needed are short-term and long-term therapeuticstrategies that have increased effectiveness in maintaining ketosis toimprove anxiety-related behavior. However, in view of the art consideredas a whole at the time the present invention was made, it was notobvious to those of ordinary skill in the field of this invention howthe shortcomings of the prior art could be overcome.

While certain aspects of conventional technologies have been discussedto facilitate disclosure of the invention, Applicants in no way disclaimthese technical aspects, and it is contemplated that the claimedinvention may encompass one or more of the conventional technicalaspects discussed herein.

The present invention may address one or more of the problems anddeficiencies of the prior art discussed above. However, it iscontemplated that the invention may prove useful in addressing otherproblems and deficiencies in a number of technical areas. Therefore, theclaimed invention should not necessarily be construed as limited toaddressing any of the particular problems or deficiencies discussedherein.

In this specification, where a document, act or item of knowledge isreferred to or discussed, this reference or discussion is not anadmission that the document, act or item of knowledge or any combinationthereof was at the priority date, publicly available, known to thepublic, part of common general knowledge, or otherwise constitutes priorart under the applicable statutory provisions; or is known to berelevant to an attempt to solve any problem with which thisspecification is concerned.

BRIEF SUMMARY OF THE INVENTION

The long-standing but heretofore unfulfilled need for an improvedtreatment for anxiety disorders and anxiety-related behaviors is now metby a new, useful, and nonobvious invention.

In an embodiment, the current invention is a method of treating apatient suffering from an anxiety disorder or a method of reducinganxiety in a patient/subject. The method comprises administering to thepatient a therapeutically effective amount of exogenous ketonesupplementation chronically or sub-chronically. The ketonesupplementation can be a ketone ester (e.g., 1,3-butanediol-acetoacetatediester), a ketone salt (e.g., beta-hydroxybutyrate-mineral salt(chronic administration)), or combined with MCT (medium chaintriglyceride), a combination thereof. Butanediol was used in aged rats.

When the ketone ester is used, it can be low concentration (˜10 g/kgb.w./day) or high concentration (˜25 g/kg b.w./day). Alternatively, whenthe ketone salt is used, it can have a concentration of ˜25 g/kgb.w./day. Optionally, the ketone salt can be admixed with a medium chaintriglyceride in a 1:1 ratio, potentially each having a concentration of˜25 g/kg b.w./day. Alternatively, sub-chronic administration canaccomplish benefits at 5 g/kg/day or 2.5 g/kg/day.

In another embodiment, the current invention is a method of reducinganxiety in a patient/subject, comprising chronically administering tothe patient or subject a therapeutically effective amount of exogenousketone supplementation. The ketone supplementation includes a ketonesalt comprising beta-hydroxybutyrate-mineral salt admixed with a mediumchain triglyceride in a 1:1 ratio, such that the concentration of thebeta-hydroxybutyrate-mineral salt and the medium chain triglyceride iseach ˜25 g/kg b.w./day.

These and other important objects, advantages, and features of theinvention will become clear as this disclosure proceeds.

The invention accordingly comprises the features of construction,combination of elements, and arrangement of parts that will beexemplified in the disclosure set forth hereinafter and the scope of theinvention will be indicated in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the invention, reference should be made tothe following detailed description, taken in connection with theaccompanying drawings, in which:

FIGS. 1A-1E depict response of SPD (Sprague-Dawley) rats to chronicfeeding of exogenous ketone supplementation. Abbreviations: SD: standardrodent chow+water (˜25 g/kg b.w. water/day); SD+LKE: SD+low-dose ketoneester (1,3-butanediol-acetoacetate diester, ˜10 g/kg b.w./day); SD+HKE:SD+high dose ketone ester (˜25 g/kg b.w./day); SD+KS:SD+beta-hydroxybutyrate-mineral salt (βHB-S) (˜25 g/kg b.w./day);SD+KSMCT: SD+βHB-S+medium chain triglyceride (MCT) (˜25 g/kg b.w./day);(*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

FIG. 1A is a graphical illustration showing that rats consuming SD+KS orSD+KSMCT supplements spent more time in open arms and less in closedarms, showing reduced anxiety compared to control (SD) group.

FIG. 1B is a graphical illustration showing that rats consuming ketonesupplements travelled more distance in open arms (SD+KS and SD-KSMCT)and less in closed arms (SD+LKE, SD+KS and SD-KSMCT), showing reducedanxiety compared to control group.

FIG. 1C is a graphical illustration showing that rats consuming SD+HKE,SD+KS or SD+KSMCT entered the closed arms later, showing reduced anxietycompared to control group.

FIG. 1D is a graphical illustration showing that rats consuming eitherketone supplements with ˜25 g/kg b.w./day dose (SD+HKE/KS/KSMCT) hadelevated blood ketone levels after 13 weeks compared to control group.

FIG. 1E is a graphical illustration showing that blood glucose levelswere be elevated in SD+KSMCT groups after 13 weeks.

FIG. 1F is a graphical illustration showing that body weight was higherin SD+LKE and SD+HKE groups, compared to control at week 13, while allbody weight increased, except SD+HKE and SD+KS, compared to baseline.

FIGS. 2A-2E depict response of SPD rats to 7 days oral administration(gavage) of exogenous ketone supplementation. Abbreviations: SD:standard rodent chow+water (˜25 g/kg b.w. water/day); KD: ketogenic diet(25 g/kg b.w./day); SD+KE: SD+ketone ester (1,3-butanediol-acetoacetatediester, ˜5 g/kg b.w./day); SD+KS: SD+beta-hydroxybutyrate-mineral salt(βHB-S) (˜5 g/kg b.w./day); SD+KSMCT: SD+βHB-S+medium chain triglyceride(MCT) (˜5 g/kg b.w./day); (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

FIG. 2A is a graphical illustration showing that less entries were incenter and in closed arms by SD+KS group.

FIG. 2B is a graphical illustration showing that more time was spent inopen arms by SD+KE group and less time spent in center by KD, SD+KE,SD+KS groups, compared to control.

FIG. 2C is a graphical illustration showing that blood βHB levels werehigher in all treatment groups after 24 hours and after 7 days as well.Specifically, blood βHB level was higher in KD group at baseline whereascompared to control, and blood βHB levels were higher in KD and SD+KEgroups.

FIG. 2D is a graphical illustration showing that blood glucose was lowerin all treatment groups, except SD+KSMCT, after 24 hours. Blood glucosewas lower in SD+KSMCT group after 7 days, compared to its baseline.Blood glucose was lower in KD, SD+KE, SD+KSMCT groups after 24 hours,compared to control. Blood glucose was lower in SD+KSMCT group after 7days, compared to control.

FIG. 2E is a graphical illustration showing that body weight increasedin SD and SD+KSMCT groups, compared to their baseline. Body weight waslower in KD and SD+KS groups at baseline, compared to control. Bodyweight was lower in all treatment groups after 7 days, compared tocontrol.

FIGS. 3A-3E depict response of WAG/Rij rats to 7 days oraladministration of exogenous ketone supplementation. Abbreviations: SD:standard rodent chow+water (˜2.5 g/kg b.w. water/day); SD+KE: SD+ketoneester (1,3-butanediol-acetoacetate diester, ˜2.5 g/kg b.w./day); SD+KS:SD+beta-hydroxybutyrate-mineral salt (βHB-S; ˜2.5 g/kg b.w./day);(*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

FIG. 3A is a graphical illustration showing that less entries were inclosed arms by SD+KS group, compared to control.

FIG. 3B is a graphical illustration showing that more time was spent inopen arms and less time spent in closed arms by SD+KS group, compared tocontrol.

FIG. 3C is a graphical illustration showing that blood βHB levels werehigher in both treatment groups (SD+KE/KS) after 7 days, compared tobaseline.

FIG. 3D is a graphical illustration showing that blood glucose level didnot change significantly in either group, compared to baseline levels.

FIG. 3E is a graphical illustration showing that body weight did notchange significantly in either group during the treatment period.

FIG. 4A is a graphical illustration depicting that 30 min after singledose administration of ketone supplements, the 1 year old SPD rats spentmore time in the open arms, compared to the control group.

FIG. 4B is a graphical illustration depicting that ˜40 min after singledose administration of ketone supplements and right after completion of5 min EPM test in 1 year old SPD rats, all treatment group hadsignificantly elevated βHB levels, compared to control group

FIG. 4C is a graphical illustration depicting that ˜40 min after singledose administration of ketone supplements and right after completion of5 min EPM test in 1 year old SPD rats, KE group had significantly lowerblood glucose level, compared to control group.

FIGS. 5A-5E depicts response of SPD rats to chronic feeding of exogenousketone supplementation. Abbreviations: SD: standard rodent chow+water(˜25 g/kg b.w. water/day); LKE: SD+low-dose ketone ester(1,3-butanediol-acetoacetate diester, ˜10 g/kg b.w./day); HKE: SD+highdose ketone ester (˜25 g/kg b.w./day); KS:SD+beta-hydroxybutyrate-mineral salt (βHB-S) (˜25 g/kg b.w./day); KSMCT:SD+βHB-S+medium chain triglyceride (MCT) (˜25 g/kg b.w./day); (*p<0.05;**p<0.01; ***p<0.001; ****p<0.0001)

FIG. 5A shows that rats consuming KSMCT supplements spent more time inopen arms (open), LKE, KS and KSMCT groups spent less time in closedarms (closed), showing reduced anxiety compared to control (SD) group.

FIG. 5B shows that rats consuming ketone supplements travelled moredistance in open arms (KS and KSMCT) and less in closed arms (LKE, KSand KSMCT), showing reduced anxiety compared to control group.

FIG. 5C shows that rats consuming KSMCT entered the closed arms later,evidencing reduced anxiety compared to control group.

FIG. 5D shows that rats consuming HKE, KS and KSMCT had elevated bloodketone levels after 13 weeks (after) compared to control group.

FIG. 5E shows that blood glucose levels were lower in HKE and KSMCTgroups compared to control after 13 weeks.

FIG. 5F shows that body weight was lower in HKE group after 13 weeks.

FIGS. 6A-6E depicts response of SPD rats to 7 days oral administration(gavage) of exogenous ketone supplementation. Abbreviations: SD:standard rodent chow+water (˜5 g/kg b.w. water/day); KE: SD+ketone ester(1,3-butanediol-acetoacetate diester, ˜5 g/kg b.w./day); KS:SD+beta-hydroxybutyrate-mineral salt (βHB-S) (˜5 g/kg b.w./day); KSMCT:SD+βHB-S+medium chain triglyceride (MCT) (˜5 g/kg b.w./day); (*p<0.05;**p<0.01; ***p<0.001; ****p<0.0001)

FIG. 6A depicts more time spent in open arms (open) by KE group and lesstime spent in center by KE, KS and KSMCT, compared to control.

FIG. 6B depicts less entries in closed arms (closed) by KE and KSgroups.

FIG. 6C shows that blood βHB levels were higher in KE group after 24hours and in KS and KSMCT groups after 7 days, compared to control.

FIG. 6D shows that blood glucose levels were lower in KE group after 24hours and in KSMCT group compared to baseline, control and 24 hours.

FIG. 6E depicts decreased body weights in KE and KS groups.

FIGS. 7A-7E depicts response of WAG/Rij rats to 7 days oraladministration of exogenous ketone supplementation. Abbreviations: SD:standard rodent chow+water (˜2.5 g/kg b.w. water/day); KE: SD+ketoneester (1,3-butanediol-acetoacetate diester, ˜2.5 g/kg b.w./day); KS:SD+beta-hydroxybutyrate-mineral salt (βHB-S) (˜2.5 g/kg b.w./day);KSMCT: SD+beta-hydroxybutyrate-mineral salt (BHB-S)+medium chaintriglyceride (MCT) (KSMCT; ˜2.5 g/kg b.w./day); (*p<0.05; **p<0.01;***p<0.001; ****p<0.0001)

FIG. 7A depicts more time spent in open arms (open) and less time spentin closed arms (closed) by KSMCT group, compared to control.

FIG. 7B depicts less entries in closed arms by KSMCT group and in openarms by KE group.

FIG. 7C shows that blood βHB levels were higher in all treatment groups(KE, KS and KSMCT) after 7 days, compared to baseline.

FIG. 7D shows that blood glucose levels decreased after 24 hours in KEgroup compared to baseline, but increased after 7 days compared to 24hours in KE and KSMCT group.

FIG. 7E shows that body weight did not change significantly in eithergroup.

FIG. 8A-8B depict blood βHB and glucose levels compared between the twoanimal models. (**p<0.01; ****p<0.0001)

FIG. 8A shows that blood βHB levels were higher at 7 days in WAG/Rijrats after KE and KSMCT treatment.

FIG. 8B shows that blood glucose levels were significantly lower inWAG/Rij rats, except in KSMCT group at 7 days.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In the following detailed description of the preferred embodiments,reference is made to the accompanying drawings, which form a partthereof, and within which are shown by way of illustration specificembodiments by which the invention may be practiced. It is to beunderstood that other embodiments may be utilized and structural changesmay be made without departing from the scope of the invention.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural referents unless the contentclearly dictates otherwise. As used in this specification and theappended claims, the term “or” is generally employed in its senseincluding “and/or” unless the context clearly dictates otherwise.

As used herein, “about” means approximately or nearly and in the contextof a numerical value or range set forth means 15% of the numerical. Inan embodiment, the term “about” can include traditional roundingaccording to significant figures of the numerical value. In addition,the phrase “about ‘x’ to ‘y’” includes “about ‘x’ to about ‘y’”.

In certain embodiments, the current invention includes methods oftreating or reducing anxiety-related behaviors. The methods includeadministering a therapeutically effective amount of ketonesupplementation, such as butanediol, ketone esters and/or ketone salts,their combinations or combined with MCT chronically, sub-chronically oracutely (see aged rats).

As used herein, “treat”, “treatment”, “treating”, and the like refer toacting upon a condition (e.g., anxiety-related behavior) with an agent(e.g., ketone supplementation) to affect the condition by improving oraltering it. The improvement or alteration may include an improvement insymptoms or an alteration in the physiologic pathways associated withthe condition. The aforementioned terms cover one or more treatments ofa condition in a patient (e.g., a mammal, typically a human or non-humananimal of veterinary interest), and includes: (a) reducing the risk ofoccurrence of the condition in a subject determined to be predisposed tothe condition but not yet diagnosed, (b) impeding the development of thecondition, and/or (c) relieving the condition, e.g., causing regressionof the condition and/or relieving one or more condition symptoms (e.g.,reduce anxiety-related behavior).

As used herein, the terms “prophylactically treat” or “prophylacticallytreating” refers completely or partially preventing (e.g., about 50% ormore, about 60% or more, about 70% or more, about 80% or more, about 90%or more, about 95% or more, or about 99% or more) a condition or symptomthereof and/or may be therapeutic in terms of a partial or complete cureor alleviation for a condition and/or adverse effect attributable to thecondition.

A “pharmaceutically acceptable excipient,” “pharmaceutically acceptablediluent,” “pharmaceutically acceptable carrier,” or “pharmaceuticallyacceptable adjuvant” means an excipient, diluent, carrier, and/oradjuvant that are useful in preparing a pharmaceutical composition thatare generally safe, non-toxic and neither biologically nor otherwiseundesirable, and include an excipient, diluent, carrier, and adjuvantthat are acceptable for veterinary use and/or human pharmaceutical use.“A pharmaceutically acceptable excipient, diluent, carrier and/oradjuvant” as used in the specification and claims includes one or moresuch excipients, diluents, carriers, and adjuvants.

The term “therapeutically effective amount” as used herein describesconcentrations or amounts of components such as agents which areeffective for producing an intended result, including preventing furtheranxiety-related behavior, or treating anxiety-related behavior andrelated conditions. Compositions according to the present invention maybe used to effect a favorable change in anxiety-related behavior,whether that change is an improvement, such as stopping or reversing thebehavior, reducing levels of the behavior, or improving the behavior,relieve to some extent one or more of the symptoms of the conditionbeing treated, and/or that amount that will prevent, to some extent, oneor more of the symptoms of the condition that the host being treated hasor is at risk of developing, or a complete cure of the disease orcondition treated.

The term “administration” or “administering” is used throughout thespecification to describe the process by which ketone supplementation ora composition comprising butanediol, beta-hydroxybutyrate or a ketonesupplement (e.g., ketone ester, ketone salt, MCT) as an active agent,are delivered to a patient for therapeutic purposes. Ketonesupplementation or the composition of the subject invention can beadministered a number of ways including, but not limited to, parenteral(such term referring to intravenous and intra-arterial as well as otherappropriate parenteral routes), subcutaneous, peritoneal, inhalation,vaginal, rectal, nasal, or instillation into body compartments.

Administration will often depend upon the amount of compoundadministered, the number of doses, and duration of treatment. In anembodiment, multiple doses of the agent are administered. The frequencyof administration of the agent can vary depending on any of a variety offactors, such as extent of anxiety-related behavior, and the like. Theduration of administration of the agent, e.g., the period of time overwhich the agent is administered, can vary, depending on any of a varietyof factors, including patient response, etc.

The amount of the agent contacted (e.g., administered) can varyaccording to factors such as the degree of susceptibility of theindividual, the age, sex, and weight of the individual, idiosyncraticresponses of the individual, the dosimetry, and the like. Detectablyeffective amounts of the agent of the present disclosure can also varyaccording to instrument and film-related factors. Optimization of suchfactors is well within the level of skill in the art, unless otherwisenoted.

As used herein, the term “subject,” “patient,” or “organism” includeshumans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses).Typical hosts to which an agent(s) of the present disclosure may beadministered will be mammals, particularly primates, especially humans.For veterinary applications, a wide variety of subjects will besuitable, e.g., livestock such as cattle, sheep, goats, cows, swine, andthe like; poultry such as chickens, ducks, geese, turkeys, and the like;and domesticated animals particularly pets such as dogs and cats. Fordiagnostic or research applications, a wide variety of mammals will besuitable subjects, including rodents (e.g., mice, rats, hamsters),rabbits, primates, and swine such as inbred pigs and the like.

It should be noted that ratios, concentrations, amounts, and othernumerical data may be expressed herein in a range format. It is to beunderstood that such a range format is used for convenience and brevity,and thus, should be interpreted in a flexible manner to include not onlythe numerical values explicitly recited as the limits of the range, butalso to include all the individual numerical values or sub-rangesencompassed within that range as if each numerical value and sub-rangeis explicitly recited. To illustrate, a concentration range of “about0.1% to about 5%” should be interpreted to include not only theexplicitly recited concentration of about 0.1 wt % to about 5 wt %, butalso include individual concentrations (e.g., 1%, 2%, 3%, and 4%) andthe sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within theindicated range.

Study 1

Nutritional ketosis has been proven effective for seizure disorders andother neurological disorders. GLUT1 deficiency syndrome (G1D) ischaracterized by seizures resulting from impaired glucose metabolism inthe brain. Anecdotal reports suggest that nutritional ketosis improvesbehavior and disposition in those with seizure disorders, including areduction in anxiety-related behavior. An objective of this study was todetermine the effects of ketone supplementation on anxiety-relatedbehavior in Sprague-Dawley (SPD) and WAG/Rij rats.

I. Methods

Generally, exogenous ketone supplements were tested and fed chronicallyfor 83 days and administered sub-chronically for 7 days by gavage,followed by assessment of anxiety measures on elevated plus maze (EPM)on 97 male SPD rats. The groups included standard diet (SD), ketogenicdiet (KD), and SD+ketone supplementation (SD+KS). Ketone supplementationincluded ketone ester (KE) [low-dose ketone ester (LKE)(1,3-butanediol-acetoacetate diester, ˜10 g/kg/day, SD+LKE) and highdose ketone ester (HKE) (1,3-butanediol-acetoacetate diester, ˜25g/kg/day, SD+HKE)], beta-hydroxybutyrate-mineral salt (βHB-S) (˜25g/kg/day, SD+KS), and βHB-S+medium chain triglyceride (MCT) (˜25g/kg/day, SD+KSMCT).

To extend the results and ensure that the results were not straindependent, exogenous ketone supplements were tested on both SPD rats andWistar Albino Glaxo/Rijswijk (WAG/Rij) rats for 7 days by gavage (5g/kg/day and 2.5 g/kg/day, respectively, SD+KE and SD+KS), as WAG/Rijrats show reduced activity of GABAergic system (Luhmann H J, et al.Impairment of intracortical GABAergic inhibition in a rat model ofabsence epilepsy. Epilepsy Res. 1995 September; 22(1):43-51).

As background, the WAG/Rij rat strain was originally developed as ananimal model of human absence epilepsy as the animals show spontaneousspike-wave discharges in the EEG (Coenen, A. M., et al, 2003. Geneticanimal models for absence epilepsy: a review of the WAG/Rij strain ofrats. Behav. Genet. 33, 635-655). Nevertheless, WAG/Rij rats are oftenused for investigation of different central nervous system (CNS)diseases, such as anxiety, similar to SPD rats by means of EPM (KovácsZ, et al. Facilitation of spike-wave discharge activity bylipopolysaccharides in Wistar Albino Glaxo/Rijswijk rats. Neuroscience.2006 Jun. 30; 140(2):731-42; Kovács Z, et al. Neonatal tricyclicantidepressant clomipramine treatment reduces the spike-wave dischargeactivity of the adult WAG/Rij rat. Brain Res Bull. 2012 Nov. 1;89(3-4):102-7; Kovács Z, et al. Absence epileptic activity changingeffects of non-adenosine nucleoside inosine, guanosine and uridine inWistar Albino Glaxo Rijswijk rats. Neuroscience. 2015 Aug. 6;300:593-608; Sarkisova K Y, et al. Depressive-like behavioralalterations and c-fos expression in the dopaminergic brain regions inWAG/Rij rats with genetic absence epilepsy. Behav Brain Res. 2003 Sep.15; 144(1-2):211-26; Sarkisova K Y, et al. Behavioral characteristics ofWAG/Rij rats susceptible and non-susceptible to audiogenic seizures.Behav Brain Res. 2006 Jan. 6; 166(1):9-18; Sarkisova K, et al. TheWAG/Rij strain: a genetic animal model of absence epilepsy withcomorbidity of depression. Prog Neuropsychopharmacol Biol Psychiatry.2011 Jun. 1; 35(4):854-76; Rebuli M E, et al. Impact of Low Dose OralExposure to Bisphenol A (BPA) on Juvenile and Adult Rat Exploratory andAnxiety Behavior: A CLARITY-BPA Consortium Study. Toxicol Sci. 2015 Jul.23. pii: kfv163). Thus, the focus of this study was to test anddetermine the effects of different types of ketone supplementation onanxiety-related behavior by using EPM behavioral assay in two ratstrains. Exogenous ketone supplements were fed chronically to SPD ratsand administered sub-chronically to SPD rats and WAG/Rij rats andacutely to SPD rats prior to assessment of anxiety measures.

A. Animals

Three-month old male Sprague-Dawley rats (SPD, n=97) and eight-month oldWAG/Rij (n=24) rats were used in the experiments. The animals werehoused at Department of Molecular Pharmacology and Physiology(Hyperbaric Biomedical Research Laboratory, Morsani College of Medicine,University of South Florida, Tampa Fla., USA) and the Department ofZoology (University of West Hungary, Savaria Campus, Szombathely,Hungary). Animals were kept in groups of 2-4 under standard laboratoryconditions (12:12 h light-dark cycle, light was on from 08.00 AM to08.00 PM) in air-conditioned rooms at 22±2° C.

Animal treatment and measuring procedures were performed in accordancewith the University of South Florida Institutional Animal Care and UseCommittee (IACUC) guidelines (Protocol #0006R) and with the localethical rules in accordance with the Hungarian Act of Animal Care andExperimentation (1998. XXVIII. Section 243/1998.) in conformity with theregulations for animal experimentation in the European CommunitiesCouncil Directive of 24 Nov. 1986 (86/609/EEC). All efforts were made toreduce the number of animals used.

B. Synthesis and Formulation of Ketone Precursors

Ketone ester (KE, which is 1,3-butanediol-acetoacetate diester) wassynthesized as previously described (D'Agostino, D., et al. (2013)Therapeutic ketosis with ketone ester delays central nervous systemoxygen toxicity seizures in rats. American Journal of Physiology.Regulatory, integrative and comparative physiology304(10):R829-36.doi:10.1152/ajpregu.00506.2012). Ketone salt (KS, whichis Na⁺/K⁺-βHB mineral salt) is a novel agent that was mixed into a 50%solution supplying approximately 375 mg/g of pure βHB and 125 mg/g ofNa⁺/K⁺. Accordingly, each dose of KS would equal ˜1000-1500 mg of HB,depending on the weight of the animal. Both KE and KS were developed andsynthesized in collaboration with Savind Inc. Human food grade MCT oil(˜65% caprylic triglyceride) was purchased from Now Foods (Bloomingdale,Ill., USA). KS was mixed with medium chain triglyceride (MCT) in a 1:1ratio (KSMCT) at the University of South Florida (USF, USA). Butanediolin aged rats.

C. Ketone Supplementation

i. Chronic Administration

In order to familiarize the animals to the intragastric gavage method,tap water was gavaged for 5 days before ketone supplementation. A totalof 48 male SPD rats were fed for 83 days with either standard rodentchow (SD)+water application (˜25 g/kg b.w. water/day) (SD/control group;n=9) or SD+ketone supplementation by gavage. Four treatment animalgroups included low-dose KE (˜10 g/kg b.w./day, SD+LKE group; n=10),high-dose KE (25 g/kg b.w./day, SD+HKE group; n=10), KS (˜25 g/kgb.w./day, SD+KS group; n=9), and KSMCT (˜25 g/kg b.w./day, SD+KSMCTgroup; n=10).

ii. Sub-Chronic Oral Gavage

Following the adaptation period to the intragastric gavage method, 51male SPD rats were fed sub-chronically for 7 days with SD (SD/controlgroup; n=11), KD (n=10) or gavaged daily with ketone supplements (5 g/kgb.w./day, SD+KE group: n=9, SD+KS group: n=9, SD+KSMCT group: n=10).

In addition, following the adaptation period to the intragastric gavagemethod, Wag/Rij male rats (n=24) were fed with SD and gavagedsub-chronically with ˜2.5 g/kg b.w./day water (n=8, SD/control group),KE (n=8; SD+KE group) or KS (n=8; SD+KS group) for 7 days.

iii. Acute Oral Gavage

One year old SPD rats were gavaged and EPM test was performed 30 minafter administration. Treatment groups BD, KE, KEMCT, KSMCT, KEKS.

D. Anxiety Assay

EPM (COULBOURN Instruments) was used to assess anxiety-related behaviorof the rats after 83 days of chronic feeding or after 7 days of oralgavage. EPM experiments were carried out under non-stressful conditions(in a dimly lit and quiet room) between 12.00 and 14.00 hours.

As background, EPM is a widely used behavioral assay for rodents, and ithas been validated to assess the anxiety responses of rodents (Pellow S,et al. Validation of open:closed arm entries in an elevated plus-maze asa measure of anxiety in the rat. J Neurosci Methods. 1985; 14:149-167;Walf, Alicia, et al. 2007. ‘The Use of The Elevated Plus Maze As AnAssay of Anxiety-Related Behavior in Rodents’. Nature Protocols 2 (2):322-328. doi:10.1038/nprot.2007.44). This test relies upon rodents'proclivity toward dark enclosed spaces (approach) and an unconditionedfear of heights/open spaces (avoidance) (Barnett, S A. The Rat—A Studyin Behavior. (Univ. Chicago Press, Chicago, 1975); Walf, Alicia, et al.2007. ‘The Use of The Elevated Plus Maze As An Assay of Anxiety-RelatedBehavior in Rodents’. Nature Protocols 2 (2): 322-328.doi:10.1038/nprot.2007.44). EPM is an investigation method of anxietythat is primarily sensitive to benzodiazepine-type anxiolytics (e.g.,diazepam) (Paslawski T, et al. The antidepressant drug phenelzineproduces antianxiety effects in the plus-maze and increases in rat brainGABA. Psychopharmacology (Berl). 1996 September; 127(1):19-24. PubMedPMID: 8880939). Anti-anxiety behavior (increased open arm time and/oropen arm entries) can be determined, which reflects the rodent'spreference (or lack thereof) for protected areas (e.g., closed arms) andtheir innate motivation to explore novel environments (Walf, Alicia, etal. 2007. ‘The Use of The Elevated Plus Maze As An Assay ofAnxiety-Related Behavior in Rodents’. Nature Protocols 2 (2): 322-328.doi:10.1038/nprot.2007.44). Consequently, EPM assay on SPD and WAG/Rijrats was a suitable method for investigating the effect of ketonesupplementation-evoked changes on anxiety level and anxiety-relatedbehaviors.

The rats were transferred in their home cage to the experimental room 30min prior to beginning the experiment. Briefly, rats were placed in theintersection of the four arms of the EPM, facing the open arm oppositeto where the experimenter was and their behavior was recorded for 5minutes. The amount of time spent and number of entries made on the openarms, closed arms and the center zones were recorded. Latency to entryinto the closed arms and the distance travelled in each zones was alsomeasured in chronically treated SPD rats. Notes were made on otherethological measures such as number of rears, head dips, fecal boli, andfreezing, or stretched-attend postures.

At the end of the 5-minute test the rats were removed from the maze andplaced back into their home cage. The maze was cleaned with 70% alcoholand after it with tap water and dried with paper towel between rats. Theprimary method for data collection was a video-tracking system withcomputer interface and video camera (SMART V3.0 PLATFORM, PANLAB,Harvard Apparatus, USA), to automatically collect behavioral data in SPDrats. A blinded observer was present in the testing room separated fromthe maze by a curtain, and manually collected EPM data in both SPD andWAG/Rij animals at the end of treatments.

E. Blood Analyses and Weight Measurement

In the chronic feeding study, blood 3HB and glucose levels were measured24 hours before the 1^(st) day of ketone treatments (baseline levels)and after the 13^(th) week of ketone treatment. In the 7-day oral gavagestudies, blood βHB and glucose levels were measured 24 hours before the1^(st) day of ketone treatments (baseline levels; SPD and WAG/Rij rats)and after the 1^(st) day (SPD rats) and the 7^(th) day (SPD and WAG/Rijrats) of ketone gavaging. In acute study, blood βHB and glucose levelswere measured 40 min after ketone treatment. Whole blood samples (10 μL)were taken from the saphenous vein for analysis of blood glucose (mg/dl)and βHB (mmol/l) levels with the commercially available glucose andketone monitoring system PRECISION XTRA™ (ABBOTT Laboratories, AbbottPark, Ill., USA). The PRECISION XTRA™ ketone monitoring system onlymeasures βHB blood levels; therefore, total blood ketone levels would behigher than measured.

The body weight of all animals was recorded before ketone treatmentsstarted (before) and after the last ketone treatments (after).

F. Statistics

All data are presented as the mean standard error of the mean (SEM). Theeffects of ketone supplementations on anxiety level were compared, aswell as on blood βHB and glucose levels to control or/and baselinelevels. Data analysis was performed using GraphPad PRISM version 6.0a.Results were considered significant when p<0.05. Significance wasdetermined by unpaired t-test. Blood ketone, blood glucose, and bodyweight change were compared using a two-way ANOVA with Tukey's multiplecomparisons test and unpaired t-test.

II. Results

It was determined herein that, when compared to control (no ketonesupplementation), ketone supplementation reduced anxiety in rats on EPMas measured by less entries to closed arms (sub-chronic SD+KS: SPD andWAG/Rij rats), more time spent in open arms (sub-chronic SD+KS: SPD andWAG/Rij rats; chronic SD+KS/KSMCT: SPD rats), more distance travelled inopen arms (chronic SD+KS/KSMCT: SPD rats), and delayed latency toentrance to closed arms (chronic SD+HKE/KS/KSMCT: SPD rats). The chronicand sub-chronic ketone supplements also caused significant elevation ofblood βHB levels and changed blood glucose levels. This data indicatesthat chronic and sub-chronic ketone supplementation not only elevatedblood ketone levels in both animal models, but reduced anxiety-relatedbehavior. These influences may also be highly beneficial for patientsmanaging diseases with nutritional ketosis.

A. Ketone Supplementation Reduced Anxiety on Elevated Plus Maze

i. Less Entries to Closed Arms with Ketone Supplements

Entries to the closed arms were less frequent with SD+KS treatment inboth rat models after 7 days of gavaging (FIGS. 2A and 3A; p=0.026;0.00046). SPD rats also entered fewer times to the center when treatedwith SD+KS (FIG. 2A; p=0.016) compared to control (SD) animals.

ii. More Time Spent in Open Arms with Ketone Supplements

After chronic feeding the time spent in the open arms was significantlymore in SD+KS and SD+KSMCT groups (p=0.017 and 0.022, respectively),while time spent in the closed arms was significantly less (p=0.01 and0.005) in these groups, compared to the control (SD). Time spent in thecenter was significantly more (p=0.04) in SD+KS group (FIG. 1A). Alsoseen in FIG. 1A, time spent in open arms were significantly more inSD+KS and SD+KSMCT groups (p=0.017 and 0.022, respectively), while timespent in closed arms were significantly less (p=0.01; 0.005) in thesegroups, compared to control (SD). Time spent in the center wassignificantly more (p=0.04) in SD+KS group.

After 7 days of gavaging SPD rats, the time spent in the open armsincreased in the SD+KE group, whereas time spent in the center decreasedin KD, SD+KE and SD+KS groups (FIG. 2B). In WAG/Rij rats the SD+KStreated rats spent more time in the open arms and less time in theclosed arms, whereas SD+KE treated rats spent more time in the center(FIG. 3B) compared to the control (SD) group.

iii. Delayed Latency of Entrance to Closed Arms with Ketone Supplements

The latency to first entrance of closed arms was significantly greaterin SD+HKE, SD+KS and SD+KSMCT groups after chronic feeding (p=0.037,0.034 and 0.014, respectively) (FIG. 1C). Also seen in FIG. 1C, latencyto first entrance to closed arms was significantly higher in HKE, SD+KSand SD+KSMCT groups (p=0.037; 0.035; 0.015, respectively), compared tocontrol (SD).

iv. More Distance Traveled in Open Arms with Ketone Supplements

After chronic feeding, the distance traveled in the open arms wassignificantly greater in SD+KS and SD+KSMCT groups (p=0.02 and 0.02),and distance traveled in the closed arms was significantly less inSD+LKE, SD+KS and SD+KSMCT groups (p=0.04, 0.009 and 0.007), compared tothe control (SD). Distance traveled in the center was more in SD+KSgroup (p=0.046; FIG. 1B). Also seen in FIG. 1B, distance travelled inopen arms were significantly more in SD+KS and SD+KSMCT groups (p=0.02;0.02), and distance travelled in closed arms were significantly less inLKE, SD+KS and SD+KSMCT groups (p=0.04; 0.009; 0.007), compared tocontrol (SD). Distance travelled in the center was more in SD+KS group(p=0.046).

B. Elevation of Blood/βHB Levels with Ketone Supplements

After 83 days of chronic feeding in SPD rats, blood βHB was elevated inSD+HKE, SD+KS and SD+KSMCT treatment groups (p=5,5E-05; 4,5E-05; 0,0004,respectively), compared to control (FIG. 1D, Table 1).

TABLE 1 Blood βHB (90 days) Tukey's multiple comparisons test Mean Diff.95% CI of diff. Significant? Summary Baseline: SD vs. After: SD 0.21780.01103 to 0.4245  Yes * Baseline: SD + HKE vs. After: SD 0.20780.001033 to 0.4145  Yes * Baseline: SD + KS vs. After: SD 0.2444 0.03233to 0.4566  Yes * Baseline: SD + KS vs. After: SD + LKE 0.2267 0.01992 to0.4334  Yes * Baseline: SD + KSMCT vs. After: SD 0.2078 0.001033 to0.4145  Yes * After: SD vs. After: SD + HKE −0.2978  −0.5045 to −0.09103Yes *** After: SD vs. After: SD + KS −0.3653 −0.5839 to −0.1466 Yes ****After: SD vs. After: SD + KSMCT −0.2778  −0.4845 to −0.07103 Yes **After: SD + LKE vs. After: SD + HKE −0.28  −0.4812 to −0.07877 Yes ***After: SD + LKE vs. After: SD + KS −0.3475 −0.5609 to −0.1341 Yes ****After: SD + LKE vs. After: SD + KSMCT −0.26  −0.4612 to −0.05877 Yes **Blood Glucose (90 days) Baseline: SD + HKE vs. After: SD + LKE −17.3 −33.96 to −0.6428 Yes * Baseline: SD + KS vs. After: SD + LKE −22.24−39.36 to −5.131 Yes ** Baseline: SD + KS vs. After: SD + KSMCT −18.24−35.36 to −1.131 Yes * Baseline: SD + KSMCT vs. After: SD + LKE −18.1−34.76 to −1.443 Yes * Body Weight (90 days) Baseline: SD vs. After: SD−95.7 −167.2 to −24.19 Yes ** Baseline: SD vs. After: SD + KSMCT −97.41−167.0 to −27.81 Yes *** Baseline: SD + LKE vs. After: SD −97.81 −169.3to −26.30 Yes ** Baseline: SD + LKE vs. After: SD + KSMCT −99.52 −169.1to −29.92 Yes *** Baseline: SD + HKE vs. After: SD −88.9 −160.4 to−17.39 Yes ** Baseline: SD + HKE vs. After: SD + KSMCT −90.61 −160.2 to−21.01 Yes ** Baseline: SD + KS vs. After: SD −84.77 −156.3 to −13.26Yes ** Baseline: SD + KS vs. After: SD + KSMCT −86.48 −156.1 to −16.88Yes ** Baseline: SD + KSMCT vs. After: SD −92.65 −164.2 to −21.14 Yes **Baseline: SD + KSMCT vs. After: SD + KSMCT −94.36 −164.0 to −24.76 Yes** After: SD vs. After: SD + HKE 73.96 2.453 to 145.5 Yes * After: SD +HKE vs. After: SD + KSMCT −75.67 −145.3 to −6.071 Yes *

Blood βHB levels were elevated in SPD rats after 24 hours of a singlegavage in KD and SD+KE groups (FIG. 2C; p=1.67E-06; 0.00015), comparedto baseline. The levels were also elevated in all treatment groups after7 days of administration, compared to the control (SD) group (FIG. 2C,Table 2).

TABLE 2 Blood βHB (7 days) Tukey's multiple comparisons test Mean Diff.95% CI of diff. Significant? Summary Baseline: SD vs. 24 h: KD −0.7182  −1.221 to −0.2153 Yes *** Baseline: SD vs. 7 days: KD −0.68   −1.196to −0.1641 Yes ** Baseline: SD vs. 7 days: SD + KSMCT −2.018   −2.521 to−1.515 Yes **** Baseline: KD vs. 24 h: KD −0.5273    −1.041 to −0.01354Yes * Baseline: KD vs. 7 days: SD + KSMCT −1.827   −2.341 to −1.314 Yes**** Baseline: SD + KE vs. 24 h: KD −0.7348   −1.238 to −0.2319 Yes ***Baseline: SD + KE vs. 7 days: KD −0.6967   −1.213 to −0.1808 Yes ***Baseline: SD + KE vs. 7 days: SD + KSMCT −2.035   −2.538 to −1.532 Yes**** Baseline: SD + KS vs. 24 h: KD −0.7   −1.214 to −0.1863 Yes ***Baseline: SD + KS vs. 7 days: KD −0.6618   −1.188 to −0.1354 Yes **Baseline: SD + KS vs. 7 days: SD + KSMCT −2   −2.514 to −1.486 Yes ****Baseline: SD + KSMCT vs. 24 h: KD −0.6682   −1.171 to −0.1653 Yes ***Baseline: SD + KSMCT vs. 7 days: KD −0.63   −1.146 to −0.1141 Yes **Baseline: SD + KSMCT vs. 7 days: SD + KSMCT −1.968   −2.471 to −1.465Yes **** 24 h: SD vs. 24 h: KD −0.8   −1.314 to −0.2863 Yes **** 24 h:SD vs. 24 h: SD + KE −0.5235    −1.026 to −0.02057 Yes * 24 h: SD vs. 7days: KD −0.7618   −1.288 to −0.2354 Yes *** 24 h: SD vs. 7 days: SD +KS −0.5455    −1.059 to −0.03172 Yes 24 h: SD vs. 7 days: SD + KSMCT−2.1   −2.614 to −1.586 Yes **** 24 h: KD vs. 24 h: SD + KS 0.7098 0.2069 to 1.213 Yes *** 24 h: KD vs. 24 h: SD + KSMCT 0.6348  0.1319 to1.138 Yes ** 24 h: KD vs. 7 days: SD 0.8015  0.2986 to 1.304 Yes **** 24h: KD vs. 7 days: SD + KE 0.596  0.05444 to 1.137 Yes * 24 h: KD vs. 7days: SD + KSMCT −1.3   −1.814 to −0.7863 Yes **** 24 h: SD + KE vs. 7days: SD 0.525  0.03314 to 1.017 Yes * 24 h: SD + KE vs. 7 days: SD +KSMCT −1.577   −2.079 to −1.074 Yes **** 24 h: SD + KS vs. 7 days: KD−0.6717   −1.188 to −0.1558 Yes ** 24 h: SD + KS vs. 7 days: SD + KSMCT−2.01   −2.513 to −1.507 Yes **** 24 h: SD + KSMCT vs. 7 days: KD−0.5967    −1.113 to −0.08080 Yes ** 24 h: SD + KSMCT vs. 7 days: SD +KSMCT −1.935   −2.438 to −1.432 Yes **** 7 days: SD vs. 7 days: KD−0.7633   −1.279 to −0.2475 Yes **** 7 days: SD vs. 7 days: SD + KS−0.547    −1.050 to −0.04406 Yes * 7 days: SD vs. 7 days: SD + KSMCT−2.102   −2.604 to −1.599 Yes **** 7 days: KD vs. 7 days: SD + KE 0.55780.004209 to 1.111 Yes * 7 days: KD vs. 7 days: SD + KSMCT −1.338  −1.865 to −0.8118 Yes **** 7 days: SD + KE vs. 7 days: SD + KSMCT−1.896   −2.437 to −1.354 Yes **** 7 days: SD + KS vs. 7 days: SD +KSMCT −1.555   −2.068 to −1.041 Yes **** Blood Glucose (7 days)Baseline: SD vs. 24 h: KD 20.45   2.876 to 38.03 Yes ** Baseline: SD vs.24 h: SD + KE 17.64   0.05754 to 35.22  Yes * Baseline: SD vs. 7 days:SD + KSMCT 28.55   10.97 to 46.12 Yes **** Baseline: KD vs. 24 h: SD−18.74   −35.95 to −1.534 Yes * Baseline: KD vs. 7 days: SD + KSMCT25.27   7.694 to 42.85 Yes *** Baseline: SD + KE vs. 24 h: KD 24   6.421to 41.58 Yes *** Baseline: SD + KE vs. 24 h: SD + KE 21.18   3.603 to38.76 Yes ** Baseline: SD + KE vs. 7 days: SD + KSMCT 32.09   14.51 to49.67 Yes **** Baseline: SD + KS vs. 24 h: SD −19.25   −36.08 to −2.420Yes * Baseline: SD + KS vs. 7 days: SD + KSMCT 24.77   7.556 to 41.97Yes *** Baseline: SD + KSMCT vs. 24 h: KD 27.09   9.512 to 44.67 Yes**** Baseline: SD + KSMCT vs. 24 h: SD + KE 24.27   6.694 to 41.85 Yes*** Baseline: SD + KSMCT vs. 7 days: SD + KSMCT 35.18   17.60 to 52.76Yes **** 24 h: SD vs. 24 h: KD 35.92   18.72 to 53.13 Yes **** 24 h: SDvs. 24 h: SD + KE 33.11   15.90 to 50.31 Yes **** 24 h: SD vs. 7 days:KD 19.83   2.181 to 37.49 Yes * 24 h: SD vs. 7 days: SD + KS 18.92  1.716 to 36.13 Yes * 24 h: SD vs. 7 days: SD + KSMCT 44.02   26.81 to61.22 Yes **** 24 h: KD vs. 24 h: SD + KS −29   −46.58 to −11.42 Yes**** 24 h: KD vs. 24 h: SD + KSMCT −25.45   −43.03 to −7.876 Yes *** 24h: KD vs. 7 days: SD −24.09   −41.30 to −6.882 Yes *** 24 h: KD vs. 7days: SD + KE −25.84   −45.00 to −6.685 Yes *** 24 h: SD + KE vs. 24 h:SD + KS −26.18   −43.76 to −8.603 Yes **** 24 h: SD + KE vs. 24 h: SD +KSMCT −22.64   −40.22 to −5.058 Yes ** 24 h: SD + KE vs. 7 days: SD−21.27   −38.48 to −4.064 Yes ** 24 h: SD + KE vs. 7 days: SD + KE−23.02   −42.18 to −3.867 Yes ** 24 h: SD + KS vs. 7 days: SD + KSMCT37.09   19.51 to 54.67 Yes **** 24 h: SD + KSMCT vs. 7 days: SD + KSMCT33.55   15.97 to 51.12 Yes **** 7 days: SD vs. 7 days: SD + KSMCT 32.18  14.97 to 49.39 Yes **** 7 days: KD vs. 7 days: SD + KSMCT 24.18  6.169 to 42.19 Yes *** 7 days: SD + KE vs. 7 days: SD + KSMCT 33.93  14.78 to 53.09 Yes **** 7 days: SD + KS vs. 7 days: SD + KSMCT 25.09  7.512 to 42.67 Yes *** Before: SD vs. After: SD −25.58   −49.81 to−1.357 Yes * Before: KD vs. After: SD −42.92   −67.14 to −18.69 Yes ****Before KD vs. After: SD + KSMCT −27.39   −52.16 to −2.623 Yes * Before:SD + KE vs. After: SD −33.75   −57.98 to −9.524 Yes *** Before: SD + KSvs. After: SD −40.83   −65.06 to −16.61 Yes **** Before: SD + KS vs.After: SD + KSMCT −25.31    −50.08 to −0.5401 Yes * Before: SD + KSMCTvs. After: SD −29.17   −53.39 to −4.941 Yes ** After: SD vs. After: KD38.74   13.33 to 64.15 Yes *** After: SD vs. After: SD + KE 37.03  10.86 to 63.19 Yes *** After: SD vs. After: SD + KS 48.25   23.48 to73.02 Yes **** After: SD + KS vs. After: SD + KSMCT −32.73   −58.03 to−7.424 Yes **

After 7 days of gavaging, blood βHB was elevated in SD+KE, SD+KS groupsin WAG/Rij rats (FIG. 3C) compared to baseline. In SPD rats, blood βHBwas also elevated in all treatment groups compared to baseline (exceptKD group) and compared to control group as well (FIG. 2C, Table 2).

C. Ketone Supplementations and Blood Glucose Levels

In SPD rats after chronic and sub-chronic ketone treatments, bloodglucose levels were lower at 24 hours in SD, KD, SD+KS, SD+KE groupscompared to baseline, and were lower in KD, SD+KE and SD+KSMCT groupscompared to the control group (FIG. 2D).

In SPD rats after 7 days of oral gavaging, blood glucose was lower inSD+KSMCT compared to control and to baseline (FIG. 2D).

After 13 weeks, blood glucose increased in SD+KSMCT group compared tothe baseline (FIG. 1D).

D. Differences in Changes of Blood Ketone and Glucose Levels Between theTwo Animal Models

There was significant difference in both βHB and glucose levels betweenthe two animal models at baseline (Table 3). While the blood glucose andβHB levels in SPD SD+KE group did not change from baseline to 7 days,there was a significant difference in blood βHB between baseline and 7days in SPD SD+KS, WAG/Rij SD+KE and WAG/Rij SD+KS groups. After 7 days,there was a significant difference in βHB between SPD SD+KE and SPDSD+KS groups, but not between WAG/Rij SD+KE and WAG/Rij SD+KS groups norwas there a difference in their glucose levels. Both βHB and glucoselevels were significantly different between SPD and WAG/Rij animals at 7days (Table 3).

TABLE 3 Compare S-D to WagRij Table Analyzed βHB Two-way ANOVA OrdinaryAlpha 0.05 % of total P value Source of Variation variation P valuesummary Significant? Interaction 4.552 0.0005 *** Yes Row Factor 25.48<0.0001 **** Yes Column Factor 54.75 <0.0001 **** Yes ANOVA table SS DFMS F (DFn, DFd) P value Interaction 0.6132 3 0.2044 F (3, 67) = P =0.0005 6.708 Row Factor 3.433 1 3.433 F (1, 67) = P < 0.0001 112.7Column Factor 7.375 3 2.458 F (3, 67) = P < 0.0001 80.68 Residual 2.04267 0.03047 Number of missing values 21 Compare cell means regardless ofrows and columns Number of families 1 Number of comparisons per family28 Alpha 0.05 Tukey's multiple comparisons test Mean Diff. 95% CI ofdiff. Significant? Summary Baseline: S-D (SD + KE) vs. Baseline: S-D−0.03485 −0.2628 to 0.1931  No ns (SD + KS) Baseline: S-D (SD + KE) vs.Baseline: WR −0.4917 −0.7409 to −0.2424 Yes **** (SD + KE) Baseline: S-D(SD + KE) vs. Baseline: WR −0.5042 −0.7534 to −0.2549 Yes **** (SD + KS)Baseline: S-D (SD + KE) vs. 7 days: S-D −0.1389 −0.3797 to 0.1019  No ns(SD + KE) Baseline: S-D (SD + KE) vs. 7 days: S-D −0.4803 −0.7083 to−0.2523 Yes **** (SD + KS) Baseline: S-D (SD + KE) vs. 7 days: WR −1.104 −1.353 to −0.8549 Yes **** (SD + KE) Baseline: S-D (SD + KE) vs. 7days: WR −1.042  −1.291 to −0.7924 Yes **** (SD + KS) Baseline: S-D(SD + KS) vs. Baseline: WR −0.4568 −0.7106 to −0.2030 Yes **** (SD + KE)Baseline: S-D (SD + KS) vs. Baseline: WR −0.4693 −0.7231 to −0.2155 Yes**** (SD + KS) Baseline: S-D (SD + KS) vs. 7 days: S-D −0.104 −0.3495 to0.1414  No ns (SD + KE) Baseline: S-D (SD + KS) vs. 7 days: S-D −0.4455−0.6783 to −0.2126 Yes **** (SD + KS) Baseline: S-D (SD + KS) vs. 7days: WR −1.069  −1.323 to −0.8155 Yes **** (SD + KE) Baseline: S-D(SD + KS) vs. 7 days: WR −1.007  −1.261 to −0.7530 Yes **** (SD + KS)Baseline: WR (SD + KE) vs. Baseline: WR −0.0125 −0.2856 to 0.2606  No ns(SD + KS) Baseline: WR (SD + KE) vs. 7 days: S-D 0.3528 0.08740 to0.6182  Yes ** (SD + KE) Baseline: WR (SD + KE) vs. 7 days: S-D 0.01136−0.2424 to 0.2651  No ns (SD + KS) Baseline: WR (SD + KE) vs. 7 days: WR−0.6125 −0.8856 to −0.3394 Yes **** (SD + KE) Baseline: WR (SD + KE) vs.7 days: WR −0.55 −0.8231 to −0.2769 Yes **** (SD + KS) Baseline: WR(SD + KS) vs. 7 days: S-D 0.3653 0.09990 to 0.6307  Yes ** (SD + KE)Baseline: WR (SD + KS) vs. 7 days: S-D 0.02386 −0.2299 to 0.2776  No ns(SD + KS) Baseline: WR (SD + KS) vs. 7 days: WR −0.6 −0.8731 to −0.3269Yes **** (SD + KE) Baseline: WR (SD + KS) vs. 7 days: WR −0.5375 −0.8106to −0.2644 Yes **** (SD + KS) 7 days: S-D (SD + KE) vs. 7 days: S-D−0.3414  −0.5869 to −0.09594 Yes ** (SD + KS) 7 days: S-D (SD + KE) vs.7 days: WR −0.9653  −1.231 to −0.6999 Yes **** (SD + KE) 7 days: S-D(SD + KE) vs. 7 days: WR −0.9028  −1.168 to −0.6374 Yes **** (SD + KS) 7days: S-D (SD + KS) vs. 7 days: WR −0.6239 −0.8776 to −0.3701 Yes ****(SD + KE) 7 days: S-D (SD + KS) vs. 7 days: WR −0.5614 −0.8151 to−0.3076 Yes **** (SD + KS) 7 days: WR (SD + KE) vs. 7 days: WR 0.0625−0.2106 to 0.3356  No ns (SD + KS) Table Analyzed Glucose Two-way ANOVAOrdinary Alpha 0.05 % of total P value Source of Variation variation Pvalue summary Significant? Interaction 0.6162 0.7389 ns No Row Factor0.9674 0.1641 ns No Column Factor 66.21 <0.0001 **** Yes ANOVA table SSDF MS F (DFn, DFd) P value Interaction 93.28 3 31.09 F (3, 66) = P =0.7389 0.4205 Row Factor 146.4 1 146.4 F (1, 66) = P = 0.1641 1.980Column Factor 10022 3 3341 F (3, 66) = P < 0.0001 45.18 Residual 4880 6673.95 Number of missing values 22 Compare cell means regardless of rowsand columns Number of families 1 Number of comparisons per family 28Alpha 0.05 Tukey's multiple comparisons test Mean Diff. 95% CI of diff.Significant? Summary Baseline: S-D (SD + KE) vs. Baseline: S-D 7.326−3.910 to 18.56 No ns (SD + KS) Baseline: S-D (SD + KE) vs. Baseline: WR28.41  15.90 to 40.92 Yes **** (SD + KE) Baseline: S-D (SD + KE) vs.Baseline: WR 28.78  16.28 to 41.29 Yes **** (SD + KS) Baseline: S-D(SD + KE) vs. 7 days: S-D −1.841 −14.35 to 10.67 No ns (SD + KE)Baseline: S-D (SD + KE) vs. 7 days: S-D 7 −4.478 to 18.48 No ns (SD +KS) Baseline: S-D (SD + KE) vs. 7 days: WR 25.66  13.15 to 38.17 Yes**** (SD + KE) Baseline: S-D (SD + KE) vs. 7 days: WR 22.28  9.777 to34.79 Yes **** (SD + KS) Baseline: S-D (SD + KS) vs. Baseline: WR 21.08 8.797 to 33.37 Yes **** (SD + KE) Baseline: S-D (SD + KS) vs. Baseline:WR 21.46  9.172 to 33.74 Yes **** (SD + KS) Baseline: S-D (SD + KS) vs.7 days: S-D −9.167 −21.45 to 3.119 No ns (SD + KE) Baseline: S-D (SD +KS) vs. 7 days: S-D −0.3258 −11.56 to 10.91 No ns (SD + KS) Baseline:S-D (SD + KS) vs. 7 days: WR 18.33  6.047 to 30.62 Yes *** (SD + KE)Baseline: S-D (SD + KS) vs. 7 days: WR 14.96  2.672 to 27.24 Yes **(SD + KS) Baseline: WR (SD + KE) vs. Baseline: WR 0.375 −13.08 to 13.83No ns (SD + KS) Baseline: WR (SD + KE) vs. 7 days: S-D −30.25  −43.71 to−16.79 Yes **** (SD + KE) Baseline: WR (SD + KE) vs. 7 days: S-D −21.41 −33.92 to −8.902 Yes **** (SD + KS) Baseline: WR (SD + KE) vs. 7 days:WR −2.75 −16.21 to 10.71 No ns (SD + KE) Baseline: WR (SD + KE) vs. 7days: WR −6.125 −19.58 to 7.334 No ns (SD + KS) Baseline: WR (SD + KS)vs. 7 days: S-D −30.63  −44.08 to −17.17 Yes **** (SD + KE) Baseline: WR(SD + KS) vs. 7 days: S-D −21.78  −34.29 to −9.277 Yes **** (SD + KS)Baseline: WR (SD + KS) vs. 7 days: WR −3.125 −16.58 to 10.33 No ns (SD +KE) Baseline: WR (SD + KS) vs. 7 days: WR −6.5 −19.96 to 6.959 No ns(SD + KS) 7 days: S-D (SD + KE) vs. 7 days: S-D 8.841 −3.667 to 21.35 Nons (SD + KS) 7 days: S-D (SD + KE) vs. 7 days: WR 27.5  14.04 to 40.96Yes **** (SD + KE) 7 days: S-D (SD + KE) vs. 7 days: WR 24.13  10.67 to37.58 Yes **** (SD + KS) 7 days: S-D (SD + KS) vs. 7 days: WR 18.66 6.152 to 31.17 Yes *** (SD + KE) 7 days: S-D (SD + KS) vs. 7 days: WR15.28  2.777 to 27.79 Yes ** (SD + KS) 7 days: WR (SD + KE) vs. 7 days:WR −3.375 −16.83 to 10.08 No ns (SD + KS)

E. Body Weight Changes During Ketone Supplementation

After chronic feeding, the body weight of SPD rats was lower in SD+HKEgroup compared to the control. The body weight increased in SD andSD+KSMCT groups, compared to their baseline (FIG. 1F, Table 1). After 7days of treatment the body weight of SPD rats increased in SD group,compared to its baseline. The body weight was lower in all treatmentgroups except SD+KSMCT after 7 days compared to control (FIG. 2E, Table2). In WAG/Rij rats the body weight did not change significantly ineither group during the treatment period (FIG. 3E).

With these data and results obtained, it can be seen that the currentstudy was able to demonstrate the anxiolytic effect of chronic andsub-chronic ketone supplementation in both SPD and WAG/Rij rats by meansof EPM, as measured by less entries to closed arms, more time spent inopen arms, more distance travelled in open arms, and delayed latency toentrance to closed arms were measured. The exogenous ketonesupplementation tested in the present study increased the open armsexploration in the EPM and increased the latency to enter the closedarms, showing anxiolytic effect

Differences could be observed between the effects of different ketonesupplements on anxiety. After 7 days treatment, KS reduced the number ofentries to the closed arms in both animal models, and it also reducedthe time spent in closed arms and increased the time spent in open armsin WAG/Rij rats. The KE increased the time spent in the open arms by SPDanimals. After 83 days treatment in SPD animals, both the KS and KSMCTtreated rats decreased time spent in the closed arms and increased timespent in the open arms and the center (KS only), increased the distancein open arms, decreased the distance in closed arms, and increased thelatency to first entry to closed arms. The LKE reduced the distance inclosed arms, and the HKE increased the latency to first entrance toclosed arms in SPD animals.

In summary, the KS seemed to be the most efficient compound to reduceanxiety together with KSMCT, but low- and high-dose KE also had abeneficial effect on reducing anxiety. Thus, theoretically, as ketonesupplementation may generate similar changes in brain neurotransmittersystems as KD by means of ketosis (FIGS. 1A-1F, 2A-2E, and 3A-3E),chronic and sub-chronic ketone supplementation-provoked anxiolyticeffects may be evoked by means of glutamatergic and/or GABAergic as wellas adenosinergic system in SPD and WAG/Rij rats. However, the knowledgeis not sufficient in the art to explain the mechanism(s) by which ketonesupplementation exerts its anti-anxiety effects. Thus, unexpectedresults were achieved herein when administering ketone supplementationto the rats.

Higher βHB levels were measured before (baseline) and after sub-chronicketone supplementation in WAG/Rij rats (SD+KE and SD+KS) compared to SPDrats (FIGS. 2C and 3C). This result and the reduced activity ofGABAergic system in WAG/Rij rat brain (Luhmann H J, et al. Impairment ofintracortical GABAergic inhibition in a rat model of absence epilepsy.Epilepsy Res. 1995 September; 22(1):43-51) may explain that 10 timeslower doses of KE and KS (˜2.5 g/kg) than applied in SPD rats (˜25 g/kg)effectively decreased the anxiety level in WAG/Rij rats. Higher basalketone levels (and its putative anti-anxiety effects) in WAG/Rij ratsmay also cause lower basal anxiety levels compared to SPD rats (e.g.,WAG/Rij rats spent more time in the open arms compared to SPD ratsbefore ketone supplements) (FIGS. 2B and 3B).

In conclusion, based on the present study, it can be concluded thatchronic and sub-chronic administration of exogenous ketonesupplementation may be an effective way to reduce anxiety. Achievingnutritional ketosis with exogenous ketone supplementation whilemaintaining a normal diet might be an alternative to the KD, or mayfurther augment the therapeutic efficacy of the KD. These elevatedketone levels have proven to be effective for alleviating symptoms ofGLUT1D syndrome, including behavioral characteristics. The foregoingdata and results show that chronic, sub-chronic and acute administrationof ketone supplements not only elevated blood ketone levels, but alsoreduced anxiety-related behavior, which can be highly beneficial forpatients managing diseases (e.g., GLUT1D syndrome) with nutritionalketosis. Since achieving nutritional ketosis requires strict dietaryrestrictions, compliance is a major difficulty in this treatment. Theadministration of exogenous ketone supplements that increase ketonelevels in the blood without dietary restrictions may be an effectiveoption to those willing to reach and stay at the state of nutritionalketosis. Exogenous ketone supplementation provides an alternative methodto reduce anxiety for healthy individuals, as well as those who need tomaintain nutritional ketosis.

Study 2

Another study was carried out using substantially similar methodologiesand procedures as in Study 1, specifically using the EPM test toevaluate anxiety-related behavior in 1-year old SD rats whenadministered ketone supplementation versus control. FIG. 4A shows that30 min after single dose administration of ketone supplements, the1-year old SD rats spent more time in open arms, compared to the controlgroup. Additionally, FIG. 4B shows that ˜40 min after single doseadministration of ketone supplements and right after completion of 5 minEPM test, all treatment group had significantly elevated βHB levels,compared to the control group. Finally, FIG. 4C shows that ˜40 min aftersingle dose administration of ketone supplements and right aftercompletion of 5 min EPM test, the KE group had significantly lower bloodglucose level, compared to the control group.

Study 3

It was an object of the current study to determine the effects of ketonesupplementation on anxiety-related behavior in SPD and WAG/Rij rats. Itwas speculated that the ketone supplements reduced anxiety-relatedbehavior not solely by elevating blood ketone levels, but also byeffecting the regulation of 5-HT levels. Studies have shown that ratspre-treated with amphetamine exhibited increased anxiety-like behavioron the EPM, which was successfully reversed by paroxetine, a selectiveserotonin (5-HT) reuptake inhibitor (Tu W, et al. Serotonin in theventral hippocampus modulates anxiety-like behavior during amphetaminewithdrawal. Neuroscience. 2014 Sep. 18; 281C:35-43. doi:10.1016/j.neuroscience.2014.09.019). Those results suggested that 5-HTlevels in the ventral hippocampus are critical for regulating anxietybehavior and that increasing 5-HT levels may be an effective strategyfor reducing anxiety (Tu W, et al. Serotonin in the ventral hippocampusmodulates anxiety-like behavior during amphetamine withdrawal.Neuroscience. 2014 Sep. 18; 281C:35-43. doi:10.1016/j.neuroscience.2014.09.019).

Generally, exogenous ketone supplementation was tested by adding to foodand feeding chronically for 83 days in SPD rats and administeringsub-chronically for 7 days in both rat models by daily intragastricgavage bolus, followed by assessment of anxiety measures on EPM. Thegroups included standard diet (SD) or SD+ketone supplementation.Low-dose ketone ester (LKE) (1,3-butanediol-acetoacetate diester, ˜10g/kg/day, LKE), high dose ketone ester (HKE) (˜25 g/kg/day, HKE),beta-hydroxybutyrate-mineral salt (βHB-S) (˜25 g/kg/day, KS), andβHB-S+MCT (˜25 g/kg/day, KSMCT) were used as ketone supplementation forchronic administration. To extend the results, exogenous ketonesupplements were also tested sub-chronically on SPD rats (KE, KS andKSMCT; 5 g/kg/day) and on WAG/Rij rats (KE, KS and KSMCT; 2.5 g/kg/day).

At the end of treatments behavioral data collection was conductedmanually by a blinded observer and with a video-tracking system, afterwhich blood βHB and glucose levels were measured. Ketone supplementationreduced anxiety on EPM as measured by less entries to closed arms(sub-chronic KE and KS: SPD rats and KSMCT: WAG/Rij rats), more timespent in open arms (sub-chronic KE: SPD and KSMCT: WAG/Rij rats; chronicKSMCT: SPD rats), more distance travelled in open arms (chronic KS andKSMCT: SPD rats), and by delayed latency to entrance to closed arms(chronic KSMCT: SPD rats), when compared to control. The data indicatedthat chronic and sub-chronic ketone supplementation not only elevatedblood βHB levels in both animal models, but reduced anxiety-relatedbehavior. It was concluded that ketone supplementation represents apromising anxiolytic strategy through a novel means of inducingnutritional ketosis.

I. Methods

A. Animals

Two months old male SPD (n=87) and eight months old male WAG/Rij (n=32)rats were used in the experiments. The animals were housed at Departmentof Molecular Pharmacology and Physiology (Hyperbaric Biomedical ResearchLaboratory, Morsani College of Medicine, University of South Florida,Tampa Fla., USA) and the Department of Zoology (University of WestHungary, Savaria Campus, Szombathely, Hungary). Animals were kept ingroups of 2-4 under standard laboratory conditions (12:12 h light-darkcycle, light was on from 08.00 AM to 08.00 PM) in air-conditioned roomsat 22±2° C.

Previous studies showed that the percentage of open arm entries linearlyincrease with age (Lynn D A, et al. The Ontogeny of Anxiety-LikeBehavior in Rats from Adolescence to Adulthood. DevelopmentalPsychobiology. 2010; 52(8):731-739. doi:10.1002/dev.20468) and theaging-related changes in EPM behavior are strain-specific (Ferguson S A,et al. Aging effects on elevated plus maze behavior in spontaneouslyhypertensive, Wistar-Kyoto and Sprague-Dawley male and female rats.Physiol Behav. 2005 Aug. 7; 85(5):621-8. PubMed PMID: 16043200). Thus,the rats used in the present study involved two strains and animals ofdifferent age to minimize or eliminate that variable.

Animal treatment and measuring procedures were performed in accordancewith the University of South Florida Institutional Animal Care and UseCommittee (IACUC) guidelines (Protocol #0006R) and with the localethical rules in accordance with the Hungarian Act of Animal Care andExperimentation (1998. XXVIII. Section 243/1998.) in conformity with theregulations for animal experimentation in the European CommunitiesCouncil Directive of 24 Nov. 1986 (86/609/EEC). All experiments wereapproved by the University of South Florida Institutional Animal Careand Use Committee and all efforts were made to reduce the number ofanimals used.

B. Synthesis and Formulation of Ketone Precursors

Ketone ester (KE; 1,3-butanediol-acetoacetate diester) was synthesizedas previously described (D'Agostino, D., Pilla, R., Held, H., Landon,C., Puchowicz, M., Brunengraber, H., Ari, C., Arnold, P., and Dean, J.(2013) Therapeutic ketosis with ketone ester delays central nervoussystem oxygen toxicity seizures in rats. American Journal of Physiology.Regulatory, integrative and comparative physiology 304(10):R829-36).Ketone salt (KS, which is Na⁺/K⁺-β-hydroxybutyrate mineral salt) is anovel agent that was mixed into a 50% solution supplying approximately375 mg/g of pure βHB and 125 mg/g of Na⁺/K⁺ in a 1:1 ratio. Accordingly,each dose of KS would equal ˜1000-1500 mg of HB, depending on the weightof the animal. Both KE and KS were developed and synthesized incollaboration with Savind Inc. Human food grade MCT oil (60% caprylictriglyceride/40 capric triglyceride) was purchased from Now Foods(Bloomingdale, Ill., USA). KS was mixed with medium chain triglyceride(MCT) in a 1:1 ratio (KSMCT) at the University of South Florida (USF,USA).

C. Ketone Supplementation

In order to determine the effect of different administration forms,chronic and sub-chronic administration were tested. During chronicadministration, the ketone supplementation was mixed into the regularrodent chow, which the animals had access to all day for several weeks.During sub-chronic administration, the ketone supplementation wasgavaged orally at a single time point daily for only 7 days.

i. Chronic administration

A total of 48 male SPD rats were fed for 83 days with either standardrodent chow (2018 Teklad Global 18% Protein Rodent Diet (#2018), Harlan)(SD/control; n=9) or SD+ketone supplementation. Four treatment animalgroups included low-dose KE (˜10 g/kg b.w./day, LKE; n=10), high-dose KE(˜25 g/kg b.w./day, HKE; n=10), KS (˜25 g/kg b.w./day, KS; n=9), andKSMCT (˜25 g/kg b.w./day, KSMCT; n=10). Higher dose was used for chronicadministration, as the rats were consuming food-integrated ketonesupplementation throughout the day, not at a single time point.

ii. Sub-Chronic Administration (Oral Gavage)

In order to familiarize the animals to the intragastric gavage method,water was gavaged for 5 days prior to ketone supplementation. Followingthe adaptation period to the intragastric gavage method, 39 male SPDrats were fed with SD (SD/control group; n=11), described in previousstudies (Poff et al., 2013) and gavaged daily with 5 g/kg b.w./day water(SD/control; n=11) or ketone supplements KE (n=9), KS (n=9), KSMCT(n=10) sub-chronically for 7 days.

In addition, following the adaptation period to the intragastric gavagemethod, WAG/Rij male rats (n=32) were fed with SD and gavagedsub-chronically with ˜2.5 g/kg b.w./day water (SD/control; n=8), KE(n=8), KS (n=8) or KSMCT (n=8) for 7 days. For the sub-chronic gavageadministration, the gavage dose was used that induced desired elevationof blood ketone based on the current inventors' previous studies (Keslet al. 2016).

D. Anxiety Assay

EPM (Coulbourn Instruments) was used to assess anxiety-related behaviorof the rats after 83 days of chronic feeding or after 7 days of oralgavage. EPM experiments were carried out under non-stress conditions (indimly lit and quiet room) between 12.00 and 14.00 hours.

The rats were transferred in their home cage to the experimental room 30min prior to beginning the experiment. Briefly, rats were placed in theintersection of the four arms of the EPM, facing the open arm oppositeto where the experimenter was and their behavior was recorded for 5minutes. The amount of time spent and number of entries made on the openarms, closed arms and the center zones were video recorded. Latency toentry into the closed arms and the distance travelled in each zones wasalso measured in chronically treated SPD rats. Only those behaviors arediscussed at each experimental scenario where significant difference wasfound. At the end of the 5-minute test, the rats were removed from themaze and placed back into their home cage. The maze was cleaned with 70%alcohol and after it with tap water and dried with paper towel betweenrats. The primary method for data collection was a video-tracking systemwith computer interface and video camera (SMART V3.0 PLATFORM, Panlab,Harvard Apparatus, USA), to automatically collect behavioral data in SPDrats. A blinded observer was present in the testing room separated fromthe maze by a curtain, and collected EPM data in both SPD and WAG/Rijanimals.

E. Blood Analyses and Weight Measurement

In the chronic feeding study, blood βHB and glucose levels were measured24 hours before the 1^(st) day of ketone treatments (baseline levels)and at the 13^(th) week after the EPM experiment. In the 7-day oralgavage studies, blood βHB and glucose levels were measured 24 hoursbefore the 1^(st) day of ketone treatments (baseline levels; SPD andWAG/Rij rats), 24 hours after the first gavage, and 60 min after gavageon the 7^(th) day (SPD and WAG/Rij rats). Whole blood samples (10 μL)were taken from the saphenous vein for analysis of blood glucose (mg/dl)and βHB (mmol/l) levels with the commercially available glucose andketone (βHB) monitoring system Precision Xtra™ (Abbott Laboratories,Abbott Park, Ill., USA).

The body weight of all animals was recorded before the first ketonetreatment (before) and on the last day of the ketone treatment (after).

F. Statistics

All data are presented as the mean standard error of the mean (SEM). Theeffects of ketone supplementations on anxiety-related behavior werecompared, as well as on blood βHB and glucose levels to control or/andbaseline levels. Data analysis was performed using GraphPad PRISMversion 6.0a. Results were considered significant when p<0.05.Significance was determined by one-way ANOVA with Fisher's LSD test forthe behavioral data. Blood ketone, blood glucose, and body weight changewere compared using a two-way ANOVA with Tukey's multiple comparisonstest.

II. Results

A. Ketone Supplementation Reduced Anxiety on Elevated Plus Maze

i. More Time Spent in Open Arms with Ketone Supplements

After chronic feeding of ketone supplementation in SPD rats the timespent in the open arms was significantly more in KSMCT group (p=0.0094),while time spent in the closed arms was significantly less in LKE, KSand KSMCT groups (p=0.0389, 0.0077 and 0.0019, respectively), comparedto the control (SD) in SPD rats. Time spent in the center wassignificantly more in KS group (p=0.0239) (FIG. 5A).

After 7 days of gavage administration in SPD rats (sub-chronic), thetime spent in the open arms increased in the KE group (p=0.0281),whereas time spent in the center decreased in KE, KS and KSMCT groups(p=0.0005, <0.0001 and 0.023, respectively) (FIG. 6A). In WAG/Rij ratsthe KSMCT treated rats spent more time in the open arms (p=0.0018) andless time in the closed arms (p=0.0003), whereas KE treated rats spentmore time in the center (p=0.0027), compared to the control (SD) group(FIG. 7A).

ii. Less Entries to Closed Arms with Ketone Supplements

Entries to the closed arms were less frequent with KE and KS treatment(p=0.0436 and 0.0234, respectively) in SPD and with KSMCT treatment(p=0.0014) in WAG/Rij rat models, respectively, after 7 days ofadministration (FIGS. 6B and 7B). SPD rats also entered fewer times tothe center when treated with KS (FIG. 6A; p=0.0193), compared to control(SD) animals. Conversely, WAG/Rij rats made less entries to open arms inKE treated group (p=0.0318).

iii. More Distance Traveled in Open Arms, Less in Closed Arms andDelayed Latency of Entrance to Closed Arms with Ketone Supplements

After chronic feeding in SPD rats, the distance traveled in the openarms was significantly greater in KS and KSMCT groups (p=0.036 and0.0165, respectively), and distance traveled in the closed arms wassignificantly less in LKE, KS and KSMCT groups (p=0.0252, 0.00041, and0.0032, respectively), compared to the control (SD). Distance traveledin the center was more in KS and KSMCT groups (p=0.0206 and 0.0482,respectively; FIG. 5B).

The latency to first entrance of closed arms was significantly greaterin KSMCT group after chronic feeding (p=0.0038) (FIG. 5C).

B. Elevation of Blood βHB Levels with Ketone Supplements

After 83 days of chronic feeding in SPD rats, blood HB levels remainedsignificantly elevated in HKE, KS and KSMCT groups, compared to control(p=0.0004, <0.0001, and 0.0014, respectively; FIG. 5D) while itdecreased in SD compared to baseline (p=0.0307).

Blood βHB levels were elevated in SPD rats after 24 hours of a singlegavage in KE group (p=0.0325; FIG. 6C), compared to control.

In SPD rats, βHB was elevated in KSMCT groups at 7 days compared totheir level at 24 h and baseline (p<0.0001; FIG. 6C). Blood βHB was alsoelevated in KS and KSMCT treatment groups compared to control group(p=0.0194 and <0.0001, respectively; FIG. 6C). After 7 days of gavage,blood βHB was elevated in KE, KS and KSMCT groups in WAG/Rij rats(p<0.0001) compared to baseline, 24 hours, and control (FIG. 7C).

C. Ketone Supplementation and Blood Glucose Levels

After 13 weeks of chronic feeding in SPD rats, blood glucose did notchange significantly in any groups (FIG. 5E).

However, in SPD rats, after sub-chronic ketone treatments, blood glucoselevels were lower at 24 hours in KE group compared to control (p<0.0001;FIG. 6D). After 7 days of oral gavage, blood glucose was lower in KSMCTcompared to control, to baseline, and to the level at 24 h in SPD rats(p<0.0001; FIG. 6D).

In WAG/Rij rats, the KE group had lower glucose levels after 24 h,compared to baseline levels (p=0.0064). However, after 7 days, theirlevels were elevated again, compared to the level at 24 h (p=0.0006)(FIG. 7D). Moreover, glucose levels were also elevated after 7 dayscompared to 24 h in KSMCT group (FIG. 7D).

D. Differences in Changes of Blood Ketone and Glucose Levels Between theTwo Animal Models

There was significant difference in βHB levels between the two animalmodels in KE and KSMCT groups at 7 days only (FIG. 8A). The glucoselevels were different between the two animal models in each treatmentgroups at each of the time points, except in KSMCT group at 7 days (FIG.8B).

E. Body Weight Changes During Ketone Supplementation

After chronic feeding, the body weight of SPD rats was lower in HKEgroup compared to the control (p=0.0366). The body weight increased inSD and KSMCT groups, compared to their baseline (p=0.0015, 0.0012; FIG.5F).

After 7 days of treatment, the body weight of SPD rats increased in SDgroup, compared to its baseline (p=0.0297). The body weight was lower inKE and KS treatment groups after 7 days, compared to control (p=0.0005and <0.0001, respectively; FIG. 6E). In WAG/Rij rats, the body weightdid not change significantly in either group during the treatment period(FIG. 7E).

Considering the totality of the foregoing results, the current study hasdemonstrated the anxiolytic effect of chronic (13 week) and sub-chronic(7 days) administration of several forms of ketone supplementation inboth SPD and WAG/Rij rats. The ketone supplements tested in this studyallowed for a rapid and controlled induction of physiologic ketosiswithout the need for fasting or severe dietary restrictions. Anxiolyticeffect was assessed by means of EPM and evidenced by less entries andtime spent in closed arms, more entries and time spent in open arms,more distance travelled in open arms, and delayed/increased latency toentrance into closed arms.

In spite of WAG/Rij rats showing different anxiety behavior compared toSPD rats (e.g., WAG/Rij rats spent approximately equal times in theclosed and open arms without ketone supplementation: FIGS. 6A and 7A),sub-chronic ketone supplementation was effective not only in SPD ratsbut also in WAG/Rij rats (FIGS. 6A-6B and 7A-7B). Thus, the results onWAG/Rij rats strengthened the premise of anxiolytic effect of exogenousketone supplementation found in SPD rats.

In summary, LKE decreased time spent in closed arms and reduced distancetravelled in closed arms after chronic treatment. Moreover, KE increasedtime spent in open arms, decreased time spent in center and decreasednumber of entries in closed arms after sub-chronic treatment in SPDrats. KS was proven to be effective in reducing time spent and distancetravelled in closed arms and increase distance traveled in open armsafter chronic treatment. It also reduced time spent and number ofentries to center, while decreasing number of entries in closed armsafter sub-chronic treatment in SPD rats. KSMCT effectively increasedtime spent and distance traveled in open arms and decreased time spentand distance traveled in closed arms, as well as delayed latency tofirst entrance to closed arms after chronic treatment in SPD rats. InWAG/Rij rats KSMCT successfully increased time spent in open arms anddecreased time spent and number of entries in closed arms aftersub-chronic treatment. Differences could be observed between the effectsof different ketone supplements on anxiety. These results indicate thatKS and KSMCT are the most effective after chronic treatment, while KEand KS seem to be the most effective after sub-chronic treatment in ratswithout pathology (SPD). In rats with pathology (WAG/Rij), KSMCT was themost effective treatment after sub-chronic administration.

Higher βHB levels were measured after sub-chronic ketone supplementationin WAG/Rij rats (KE and KSMCT) compared to SPD rats (FIG. 8A). Thisresult and the reduced activity of GABAergic system in WAG/Rij rat brain(Luhmann et al., 1995) may explain that half doses of KE and KSMCT (˜2.5g/kg b.w./day) than applied in SPD rats (˜5 g/kg b.w./day) effectivelydecreased the anxiety level in WAG/Rij rats. Higher basal ketone levels(and its putative anti-anxiety effects) in WAG/Rij rats may also causelower basal anxiety levels compared to SPD rats (e.g., WAG/Rij ratsspent more time in the open arms compared to SPD rats before ketonesupplements) (FIGS. 6A and 7A).

In conclusion, based on the present study, it can be concluded thatchronic and sub-chronic administration of exogenous ketonesupplementation may be an effective way to reduce anxiety.

All referenced publications are incorporated herein by reference intheir entirety. Furthermore, where a definition or use of a term in areference, which is incorporated by reference herein, is inconsistent orcontrary to the definition of that term provided herein, the definitionof that term provided herein applies and the definition of that term inthe reference does not apply.

Glossary of Claim Terms

Administer: This term is used herein to refer to the process by whichketone supplementation or a composition comprising a ketone supplement(e.g., ketone ester, ketone salt) as an active agent, are delivered to apatient for therapeutic purposes. Ketone supplementation or thecomposition of the subject invention can be administered a number ofways including, but not limited to, parenteral (such term referring tointravenous and intra-arterial as well as other appropriate parenteralroutes), subcutaneous, peritoneal, inhalation, vaginal, rectal, nasal,or instillation into body compartments. Administration will often dependupon the amount of compound administered, the number of doses, andduration of treatment. In an embodiment, multiple doses of the agent areadministered. The frequency of administration of the agent can varydepending on any of a variety of factors, such as extent ofanxiety-related behavior, and the like. The duration of administrationof the agent, e.g., the period of time over which the agent isadministered, can vary, depending on any of a variety of factors,including patient response, etc. The amount of the agent contacted(e.g., administered) can vary according to factors such as the degree ofsusceptibility of the individual, the age, sex, and weight of theindividual, idiosyncratic responses of the individual, the dosimetry,and the like. Detectably effective amounts of the agent of the presentdisclosure can also vary according to instrument and film-relatedfactors. Optimization of such factors is well within the level of skillin the art, unless otherwise noted.

Anxiety disorder: This term is used herein to refer to a disease orcondition that is characterized by inward or outward manifestations ofanxiety.

Chronically: This term is used herein to refer to long-term treatment ofan individual subject suffering from an anxiety disorder or experiencinganxiety-related behavior, by administering ketone supplementation. Inthe studies presented herein, a long-term treatment period wasconsidered to be about 83 days.

Exogenous ketone supplementation: This term is used herein to refer to acompound—including BD, ketone salt, ketone ester, or combination thereofor combined with MCT—administered to and ingested by a patient orsubject suffering from an anxiety disorder or experiencinganxiety-related behavior.

Patient: This term is used herein to refer to a human or mammal (e.g.,mouse, rat, pig, cat, dog, and horse). Typical hosts to which anagent(s) of the present disclosure may be administered will be mammals,particularly primates, especially humans. For veterinary applications, awide variety of subjects will be suitable, e.g., livestock such ascattle, sheep, goats, cows, swine, and the like; poultry such aschickens, ducks, geese, turkeys, and the like; and domesticated animalsparticularly pets such as dogs and cats. For diagnostic or researchapplications, a wide variety of mammals will be suitable subjects,including rodents (e.g., mice, rats, hamsters), rabbits, primates, andswine such as inbred pigs and the like.

Sub-chronically: This term is used herein to refer to a shorter-termtreatment of an individual subject suffering from an anxiety disorder orexperiencing anxiety-related behavior, by administering ketonesupplementation. In the studies presented herein, a short-term treatmentperiod was considered to be about 7 days. These short-term treatmentsmay be repeated, as needed.

Therapeutically effective amount: This term is used herein to refer todescribes concentrations or amounts of components such as agents whichare effective for producing an intended result, including preventingfurther anxiety-related behavior, or treating anxiety-related behaviorand related conditions. Compositions according to the present inventionmay be used to effect a favorable change in anxiety-related behavior,whether that change is an improvement, such as stopping or reversing thebehavior, reducing levels of the behavior, or improving the behavior,relieve to some extent one or more of the symptoms of the conditionbeing treated, and/or that amount that will prevent, to some extent, oneor more of the symptoms of the condition that the host being treated hasor is at risk of developing, or a complete cure of the disease orcondition treated.

Treating: This term is used herein to refer to acting upon a condition(e.g., anxiety-related behavior) with an agent (e.g., ketonesupplementation) to affect the condition by improving or altering it.The improvement or alteration may include an improvement in symptoms oran alteration in the physiologic pathways associated with the condition.The aforementioned terms cover one or more treatments of a condition ina patient (e.g., a mammal, typically a human or non-human animal ofveterinary interest), and includes: (a) reducing the risk of occurrenceof the condition in a subject determined to be predisposed to thecondition but not yet diagnosed, (b) impeding the development of thecondition, and/or (c) relieving the condition, e.g., causing regressionof the condition and/or relieving one or more condition symptoms (e.g.,reduce anxiety-related behavior).

The advantages set forth above, and those made apparent from theforegoing description, are efficiently attained. Since certain changesmay be made in the above construction without departing from the scopeof the invention, it is intended that all matters contained in theforegoing description or shown in the accompanying drawings shall beinterpreted as illustrative and not in a limiting sense.

It is also to be understood that the following claims are intended tocover all of the generic and specific features of the invention hereindescribed, and all statements of the scope of the invention that, as amatter of language, might be said to fall therebetween.

What is claimed is:
 1. A method of treating a patient suffering from ananxiety disorder, comprising orally administering to the patient atherapeutically effective amount of exogenous ketone supplementationchronically, sub-chronically, or acutely, wherein the ketonesupplementation comprises a medium chain triglyceride in combinationwith a beta hydroxybutyrate mineral salt.
 2. The method as in claim 1,wherein the beta-hydroxybutyrate mineral salt is in a concentration ofabout 0.4 to 25 grams of the salt per kilogram of body weight per day.3. The method as in claim 1, wherein the beta-hydroxybutyrate mineralsalt is admixed with the medium chain triglyceride in a 1:1 ratio. 4.The method as in claim 1, wherein the ketone supplementation isadministered chronically.
 5. A method of reducing anxiety in a patientor subject, comprising administering to the patient or subject atherapeutically effective amount of exogenous ketone supplementationchronically or sub-chronically, wherein the ketone supplementationcomprises a medium chain triglyceride in combination with a betahydroxybutyrate mineral salt.
 6. The method as in claim 5, wherein theketone supplementation includes the beta hydroxybutyrate mineral salt ina concentration of about 0.4 to 25 grams of the salt per kilogram ofbody weight per day.
 7. The method as in claim 5, wherein the betahydroxybutyrate mineral salt is admixed with the medium chaintriglyceride in a 1:1 ratio.
 8. The method as in claim 5, wherein theketone supplementation is administered chronically.
 9. A method ofreducing anxiety in a patient or subject, comprising chronicallyadministering to the patient or subject a therapeutically effectiveamount of exogenous ketone supplementation, wherein the ketonesupplementation includes a ketone salt comprising beta-hydroxybutyratesodium and/or potassium salt admixed with a medium chain triglyceride ina 1:1 ratio, such that a concentration of thebeta-hydroxybutyrate-mineral salt is about 0.4-25 grams of thebeta-hydroxybutyrate-mineral salt per kilogram of body weight per dayand a concentration of the medium chain triglyceride is about 0.4-25grams of the medium chain triglyceride per kilogram of body weight perday.
 10. The method of claim 9, wherein the anxiety disorder isassociated with conditioned fear.